Cell Biology and Genetics

Syllabus

Unit – I

Prokaryotic and eukaryotic cells. Plasma membrane: Fluid mosaic model. Transport across cell membrane: passive and active transport.  Nucleus: ultra structure and Function.  Cell division: mitosis and meiosis.

Unit - II

Ultra structure, types and special functions of RER, SER Golgi complex, Mitochondria and Lysosome.

                       

Unit - III

DNA - Watson and Crick model of double helix, different forms of double helix – A, B & Z forms. DNA replication: types, enzymology and mechanism of semi-conservative mode of replication. RNA structure and functions of rRNA, tRNA, and mRNA.       

                                   

Unit - IV

Transcription of prokaryotic and eukaryotic genes, post transcriptional modifications of mRNA. Protein synthesis: the genetic code. Steps in protein synthesis: initiation, elongation, termination and polysomes formation. Post translational modifications.

Unit - V

Mutations – spontaneous and induced mutation, Mutagenesis by nitrous acid, hydroxylamine and intercalators. DNA damage and DNA repair mechanisms.         

Unit – 1

Eukaryotic Versus Prokaryotic Cells:

1. Nuclear body

Eukaryotic cell

The nuclear body is bounded by a nuclear membrane having pores connecting it with the endoplasmic reticulum.

It contains one or more paired, linear chromosomes composed of deoxyribonucleic acid (DNA) associated with histone proteins.

A nucleolus is present. The nuclear body is called a nucleus.

Prokaryotic cell

a. The nuclear body is not bounded by a nuclear membrane.

b. It usually contains one circular chromosome composed of deoxyribonucleic acid                                                                      (DNA) associated with histone-like proteins.

c. There is no nucleolus.

d. The nuclear body is called a nucleoid.

2. Cell division

Eukaryotic cell

a. The nucleus divides by mitosis.
b. Haploid (1N) sex cells in diploid or 2N organisms are produced through meiosis.

Prokaryotic cell

a. The cell usually divides by binary fission. There is no mitosis.
b. Prokaryotic cells are haploid. Meiosis is not needed.

3. Cytoplasmic membrane - also known as a cell membrane or plasma membrane

Eukaryotic cell

a. The cytoplasmic membrane is a fluid phospholipid bilayer  containing sterols.
b. The membrane is capable of endocytosis  (phagocytosis and pinocytosis) and   exocytosis.

Prokaryotic cell

a. The cytoplasmic membrane; is a fluid phospholipid bilayer  usually lacking sterols . Many bacteria do contain sterol-like molecules called hopanoids.
b.The membrane is incapable of endocytosis and exocytosis.

4. Cytoplasmic structures

Eukaryotic cell

a. The ribosomes  are composed of a 60S and a 40S subunit forming an 80S ribosome.
b. Internal membrane-bound organelles such as mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, and lysosomes  are present .
c. Chloroplasts  serve as organelles for photosynthesis.
d. A mitotic spindle involved in mitosis is present during cell division.
e. A cytoskeleton  is present. It contains microtubules, actin micofilaments, and intermediate filaments. These collectively play a role in giving shape to cells, allowing for cell movement, movement of organelles within the cell and endocytosis, and cell division.

Prokaryotic cell

a. The ribosomes  are composed of a 50S and a 30S subunit forming an 70S ribosome.
b. Internal membrane-bound organelles such as mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, and lysosomes are absent.
b. There are no chloroplasts. Photosynthesis usually takes place in infoldings or extensions derived from the cytoplasmic membrane.
c. There is no mitosis and no mitotic spindle.
d. They may contains only actin-like proteins that, along with the cell wall, contribute to cell shape.

5. Respiratory enzymes and electron transport chains

Eukaryotic cell

- The electron transport system is located in the inner membrane of the mitochondria.

Prokaryotic cell

- The electron transport system is located in the cytoplasmic membrane.

6. Cell wall

Eukaryotic cell

a. Plant cells, algae, and fungi have cell walls, usually composed of cellulose or chitin. Eukaryotic cell walls are never composed of peptidoglycan.
b. Animal cells and protozoans lack cell walls.

Prokaryotic cell

a. With few exceptions, members of the domain Bacteria have cell walls composed of peptidoglycan  .
b. Members of the domain Archae have cell walls composed of protein, a complex carbohydrate, or unique molecules resembling but not the same as peptidoglycan.

7. Locomotor organelles

Eukaryotic cell

- Eukaryotic cells may have flagella or cilia. Flagella and cilia are organelles involved in locomotion and in eukaryotic cells consist of a distinct arrangement of sliding microtubules surrounded by a membrane. The microtubule arrangement is referred to as a 2X9+2 arrangement.

Prokaryotic cell

- Many prokaryotes have flagella, each composed of a single, rotating fibril and usually not surrounded by a membrane. There are no cilia.

8. Representative organisms

Eukaryotic cell

- The domain Eukarya: animals, plants, algae, protozoans, and fungi.

Prokaryotic cell

- The domain Bacteria and the domain Archae.

Some important Eukaryotic cell characters:

• The Nucleus – the “brain” or control center of the cell. It contains DNA, which makes up genes. That DNA gets transcribed, or copied onto messenger RNA. That messenger carries a copy of the genes orders for certain protein production. These orders go to the protein factories.
   
• Ribosomes – These are the protein factories. They follow instructions from messenger RNA (remember that the messenger RNA got its orders from the DNA). The instructions tell the ribosomes to make specific proteins. Note, this particular organelle is found in prokaryotes too!
   
• Endoplasmic Reticulum (ER) – structures that modify proteins produced in the ribosomes. Not all of the proteins made by the ribosomes need changing, but those that do get “altered” here.
   
• Golgi Apparatus – This structure will make even more changes to the proteins that already got changed when they were in the E.R. Remember those proteins were made in the ribosomes, changed once in the E.R. and will be changed again in the Golgi Apparatus. The Golgi also acts as a post office by packaging and shipping proteins to other parts of the cell or out of the cell.
   
• Mitochondria – structures which produce the cell’s energy, a.k.a. powerhouses of the cell.
   
• Chloroplasts – structures which allow plants to trap sunlight and carry out photosynthesis.

                     Eukaryotic Cell                             

                                                                             

Prokaryotic Cell

           

Plasma Membrane:

The structure of a plasma membrane, showing its lipid bilayer and embedded proteins

The cell membrane (or plasma membrane) is the thin outer layer of the cell that differentiates the cell from its environment. In animals, the cell membrane establishes this separation alone, whereas in yeast, bacteria and plants, an additional cell wall forms the outermost boundary, providing primarily mechanical support.

As a semi-permeable barrier, the cell membrane maintains an essential balance between individual distinctness and communal interaction: it functions to retain key components of the cell and to keep out toxic or unwanted substances, while selectively controlling the flow of nutrients and biochemical signals into the cell.

The cell membrane is composed mainly of phospholipid and protein molecules arranged in organized but flexible sheets. The phospholipid components form a bilayer that contributes structural stability and creates the semi-permeable environment, while the proteins are responsible for most of the dynamic processes carried out by cell membranes, such as the transport of molecules into and out of the cell.

Transport across the cell membrane underlies a variety of physiological processes, from the beating of an animal’s heart to the opening of tiny pores in leaves that enables gas exchange with the environment. A major cellular manifestation of motor neuron disease is the inability of nerve cells to stimulate the opening of channels through the membranes of muscle cells, which would result in normal muscle function.

The regulation of transport, though a crucial function of the cell membrane, is not its only role. Cell membranes assist in the organization of individual cells to form tissues. They are also involved in biological communication: the binding of a specific substance to the exterior of the membrane can initiate, modify, or turn off a cell function.

Components: 

Lipids

The phospholipid shown has a polar head group (P) and a non-polar tail (U for unpolar). This amphipathic quality enables the phospholipid components of the cell membrane to self-organize into a lipid bilayer.

The three major types of lipids found in the cell membrane are phospholipids, glycolipids, and cholesterol molecules.

A phospholipid is composed of a polar head (a negatively charged phosphate group) and two non-polar tails (its two fatty acid chains). Phospholipids are said to be amphipathic molecules because they contain a hydrophilic (water-loving) head and two hydrophobic (water-fearing) tails. This amphipathic property causes multitudes of phospholipids suspended in water to organize naturally into a spherical, three-dimensional bilayer, which, becomes the cell membrane. Two phospholipid molecules in water will tend to join together via their water-fearing tails, and billions of them will cluster together side-by-side in the same fashion until the growing sheet curves back to make a closed sphere.

The length and properties of the fatty-acid components of phospholipids determine the fluidity of the cell membrane. At reduced temperatures, some organisms may vary the type and relative amounts of lipids to maintain the fluidity of their membranes. These changes in membrane lipid components contribute to the survival of plants, bacteria, and hibernating animals during winter.

The regulation of membrane fluidity is assisted by another lipid, cholesterol, which is primarily found in eukaryotes. (In prokaryotes, hopanoids perform a similar function.)

Carbohydrate components are linked to lipids (to form glycolipids) or to proteins (glycoproteins) on the outside of the cell membrane. They are crucial in recognizing specific molecules or other cells. For example, the carbohydrate unit of some glycolipids changes when a cell becomes cancerous, which may allow white blood cells to target cancer cells for destruction.

Proteins

Cell membranes contain two types of proteins:

• Extrinsic or peripheral proteins simply adhere to the membrane and are bound by polar interactions.
• Intrinsic proteins or integral membrane proteins may be said to reside within the membrane or to span it. They interact extensively with the fatty acid chains of membrane lipids and can be released only by agents that compete for these non-polar interactions. In addition, the cytoskeleton, which undergirds the cell membrane, provides anchoring points for integral membrane proteins.

The protein components of cell membranes may function as channels or transporters across the membrane or as receptors of biochemical information.

The relative number of proteins and lipids depends on the specialized function of the cell. For example, myelin, a membrane that encloses some nerve cells, uses properties of lipids to act as an insulator, and so contains only one protein per 70 lipids. In general, most cell membranes are about 50 percent protein by weight.

The fluid-mosaic model:

The cell membrane is often described as a fluid mosaic—a two-dimensional fluid of freely diffusing lipids, dotted or embedded with proteins. The model was first proposed by S. Jonathan Singer (1971) as a lipid-protein model and extended to include the fluid character in a publication with Garth L. Nicolson in Science (1972). Proteins are free to diffuse laterally in the lipid matrix unless restricted by specific interactions, but not to rotate from one side of the membrane to another.

Rather than presenting always a formless and fluid contour, however, the surface of the cell membrane may show structure. Synapses, the junctions between nerve cells, are one example of a highly structured membrane.

Transport across the cell membrane:

As the cell membrane is semi-permeable, only some molecules can pass unhindered into or out of the cell. These molecules are typically either small or non-polar. The cell membrane has a low permeability to ions and most polar molecules, with water being a notable exception.

There are two major mechanisms for moving chemical substances across membranes: passive transport (which does not require the input of outside energy) and active transport (which is driven by the direct or indirect input of chemical energy in the form of ATP).

Passive transport

Passive transport processes rely on a concentration gradient (a difference in concentration between the two sides of the membrane). This spontaneous process acts to decrease free energy and increase entropy in a system. There are two types of passive transport:

• Simple diffusion of hydrophobic (non-polar) and small polar molecules through the phospholipid bilayer.
• Facilitated diffusion of polar and ionic molecules, which relies on a transport protein to provide a channel or bind to specific molecules. Channels form continuous polar pathways across membranes that allow ions to flow rapidly down their electrochemical gradients (i.e., in a thermodynamically favorable direction).

Active transport

Charged or polar molecules (such as amino acids, sugars, and ions) do not pass readily through the lipid bilayer. Protein pumps use a source of free energy, like ATP or light, to drive the uphill transport. That is, active transport typically moves molecules against their electrochemical gradient, a process that would be entropically unfavorable were it not coupled with the hydrolysis of ATP. This coupling can be either primary or secondary:

• Primary active transport involves the direct participation of ATP.
• In secondary active transport, energy derived from the transport of a molecule (such as sodium) in the direction of its electrochemical gradient is used to move another molecule against its gradient.

In general, active transport is much slower than pasive transport through channels.

The processes of endocytosis, which bring macromolecules, large particles, and even small cells into a eukaryotic cell, can be thought of as examples of active transport. In endocytosis, the plasma membrane folds inward around materials from the environment, forming a small pocket. The pocket deepens, forming a vesicle that separates from the membrane and migrates into the cell interior. In exocytosis, materials packaged in vesicles are exported from a cell when the vesicle membrane fuses with the cell membrane.

Other functions of cell membranes

1. Organization. Some receptors on the external surface of the cell membrane participate in the grouping of cells to form tissues (cellular adhesion).
2. Information processing. Membrane proteins may act as receptors for the various chemical messages that pass between cells. The movement of bacteria toward food and the response of target cells to hormones such as insulin are two examples of processes that hinge on the detection of a signal by a specific receptor in a cell membrane.
3. Enzyme assembly. Cell membranes can serve as an assembly that organizes the specific enzymes involved in a given metabolic pathway. Binding the enzymes to the membrane in sequential order enables the series of chemical reactions in the pathway to be carried out efficiently.
4. Biological communication. Some membranes generate chemical or electrical signals. Cell membranes of nerve cells, muscle cells, and some eggs are excitable electrically. In nerve cells, for example, the plasma membrane conducts the nerve impulse from one end of the cell to the other.

Cell Nucleus: Structure and Functions:

The nucleus is the control center of a eukaryotic cell, responsible for the coordination of genes and gene expression. The structure of a nucleus encompasses nuclear membrane, nucleoplasm, chromosomes and nucleolus.

Nucleus is a spherical-shaped organelle present in every eukaryotic cell. As compared to other cell organelles, the nucleus is the most prominent one, which accounts to about 10 percent of the cell's volume. In general, a eukaryotic cell has only one nucleus. However, some eukaryotic cells are enucleate cells (without nucleus), for example, red blood cells (RBCs); whereas, some are multinucleate (consists of two or more nuclei), for example, slime molds. Nucleus is separated from the rest of the cell or cytoplasm by a nuclear membrane.

                                   

Cell Nucleus: Structure

        The structure of a cell nucleus consists of nuclear membrane (nuclear envelope), nucleoplasm, nucleolus and chromosomes. Nucleoplasm, also known as karyoplasm, is the matrix present inside the nucleus. Let's discuss in brief about the several parts of a cell nucleus.

Nuclear Membrane

        The nuclear membrane is a double-layered structure that encloses the contents of the nucleus. The outer layer of the nuclear membrane is connected to the endoplasmic reticulum. A fluid-filled space or perinuclear space is present between the two layers of a nuclear membrane. The nucleus communicates with the remaining of the cell or cytoplasm through several openings called nuclear pores. Nuclear pores are the sites for the exchange of large molecules (proteins and RNA) between the nucleus and cytoplasm.

Chromosomes

            Chromosomes are present in the form of strings of DNA and histones (protein molecules) called chromatin. Chromatin is further classified into heterochromatin and euchromatin based on the function. The former type is a highly condensed trancriptionally inactive form, mostly present in adjacent to the nuclear membrane. Euchromatin is a delicate, less condensed organization of chromatin, which is found abundantly in a transcribing cell.

Nucleolus

            The nucleolus is a dense, spherical-shaped structure present inside the nucleus. Some of the eukaryotic organisms have nucleus that contains up to four nucleoli. The nucleolus plays an indirect role in protein synthesis by producing ribosomes. Ribosomes are cell organelles made up of RNA and proteins; they are transported to the cytoplasm, which are then attached to the endoplasmic reticulum. Ribosomes are the protein-producing structures of a cell. Nucleolus disappears when a cell undergoes division and is reformed after the completion of cell-division.

Cell Nucleus: Functions

          Speaking about the functions of a cell nucleus, it controls the hereditary characteristics of an organism and is responsible for the protein synthesis, cell division, growth and differentiation. Here is a list of the functions carried out by a cell nucleus:

• Storage of hereditary material, the genes in the form of long and thin DNA (deoxyribonucleic acid) strands, referred to as chromatins.
• Storage of proteins and RNA (ribonucleic acid) in the nucleolus.
• Nucleus is a site for transcription in which messenger RNA (MRNA) are produced for the protein synthesis.
• Exchange of hereditary molecules (DNA and RNA) between the nucleus and rest of the cell.
• During the cell division, chromatins are arranged into chromosomes.
• Production of ribosomes (protein factories) in the nucleolus.
• Selective transportation of regulatory factors and energy molecules through nuclear pores.

As the nucleus regulates the integrity of genes and gene expression, it is also referred to as the control center of a cell. Overall, the cell nucleus stores all the chromosomal DNA of an organism.

Mitosis:

Mitosis divides the chromosomes in a cell nucleus.

Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets in two nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly equal shares of these cellular components. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle - the division of the mother cell into two daughter cells, genetically identical to each other and to their parent cell. This accounts for approximately 10% of the cell cycle.

Prokaryotic cells undergo a process similar to mitosis called binary fission. However, prokaryotes cannot be properly said to undergo mitosis because they lack a nucleus and only have a single chromosome with no centromere.

Phases of cell cycle and mitosis:

Interphase

The mitotic phase is a relatively short period of the cell cycle. It alternates with the much longer interphase, where the cell prepares itself for cell division. Interphase is therefore not part of mitosis. Interphase is divided into three phases, G₁ (first gap), S (synthesis), and G₂ (second gap). During all three phases, the cell grows by producing proteins and cytoplasmic organelles. However, chromosomes are replicated only during the S phase. Thus, a cell grows (G₁), continues to grow as it duplicates its chromosomes (S), grows more and prepares for mitosis (G₂), and finally divides (M) before restarting the cycle.

Preprophase

In plant cells only, prophase is preceded by a pre-prophase stage. In highly vacuolated plant cells, the nucleus has to migrate into the center of the cell before mitosis can begin. This is achieved through the formation of a phragmosome, a transverse sheet of cytoplasm that bisects the cell along the future plane of cell division. In addition to phragmosome formation, preprophase is characterized by the formation of a ring of microtubules and actin filaments (called preprophase band) underneath the plasma membrane around the equatorial plane of the future mitotic spindle. This band marks the position where the cell will eventually divide. The cells of higher plants (such as the flowering plants) lack centrioles: with microtubules forming a spindle on the surface of the nucleus and then being organized into a spindle by the chromosomes themselves, after the nuclear membrane breaks down. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase.

                                         

Prophase: The two round objects above the nucleus are the centrosomes. The chromatin has condensed.  

Prometaphase: The nuclear membrane has degraded, and microtubules have invaded the nuclear space. These microtubules can attach to kinetochores or they can interact with opposing microtubules.  

                                                              

Metaphase: The chromosomes have aligned at the metaphase plate.  

Early anaphase: Kinetochore microtubules shorten.  

                                

Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Note cytokinesis has already begun, the pinching is known as the cleavage furrow.  

Prometaphase

The nuclear envelope disassembles and microtubules invade the nuclear space. This is called open mitosis, and it occurs in most multicellular organisms. Fungi and some protists, such as algae or trichomonads, undergo a variation called closed mitosis where the spindle forms inside the nucleus or its microtubules are able to penetrate an intact nuclear envelope.

Metaphase

A cell in late metaphase. All chromosomes (blue) but one have arrived at the metaphase plate.  As microtubules find and attach to kinetochores in prometaphase, the centromeres of the chromosomes convene along the metaphase plate or equatorial plane, an imaginary line that is equidistant from the two centrosome poles.

Anaphase

When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate, the cell proceeds to anaphase (from the Greek,  meaning “up,” “against,” “back,” or “re-”).

Two events then occur; First, the proteins that bind sister chromatids together are cleaved, allowing them to separate. These sister chromatids, which have now become distinct sister chromosomes, are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. Next, the nonkinetochore microtubules elongate, pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. The force that causes the centrosomes to move towards the ends of the cell is still unknown, although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement.

These two stages are sometimes called early and late anaphase. Early anaphase is usually defined as the separation of the sister chromatids, while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. At the end of anaphase, the cell has succeeded in separating identical copies of the genetic material into two distinct populations.

Telophase

Telophase (from the Greek, meaning "end") is a reversal of prophase and prometaphase events. It "cleans up" the after effects of mitosis. At telophase, the nonkinetochore microtubules continue to lengthen, elongating the cell even more. Corresponding sister chromosomes attach at opposite ends of the cell. A new nuclear envelope, using fragments of the parent cell's nuclear membrane, forms around each set of separated sister chromosomes. Both sets of chromosomes, now surrounded by new nuclei, unfold back into chromatin. Mitosis is complete, but cell division is not yet complete.

Cytokinesis

Cytokinesis is often mistakenly thought to be the final part of telophase; however, cytokinesis is a separate process that begins at the same time as telophase. Cytokinesis is technically not even a phase of mitosis, but rather a separate process, necessary for completing cell division. In animal cells, a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be, pinching off the separated nuclei. In both animal and plant cells, cell division is also driven by vesicles derived from the Golgi apparatus, which move along microtubules to the middle of the cell. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall, separating the two nuclei. The phragmoplast is a microtubule structure typical for higher plants, whereas some green algae use a phycoplast microtubule array during cytokinesis. Each daughter cell has a complete copy of the genome of its parent cell. The end of cytokinesis marks the end of the M-phase.

Significance

Mitosis is important for the maintenance of the chromosomal set; each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. Transcription is generally believed to cease during mitosis, but epigenetic mechanisms such as bookmarking function during this stage of the cell cycle to ensure that the "memory" of which genes were active prior to entry into mitosis are transmitted to the daughter cells.

Meiosis:

Events involving meiosis, showing chromosomal crossover

In biology, meiosis is a process of reductional division in which the number of chromosomes per cell is cut in half. In animals, meiosis always results in the formation of gametes, while in other organisms it can give rise to spores. As with mitosis, before meiosis begins, the DNA in the original cell is replicated during S-phase of the cell cycle. Two cell divisions separate the replicated chromosomes into four haploid gametes or spores.

Meiosis is essential for sexual reproduction and therefore occurs in all eukaryotes (including single-celled organisms) that reproduce sexually. A few eukaryotes, notably the Bdelloid rotifers, have lost the ability to carry out meiosis and have acquired the ability to reproduce by parthenogenesis. Meiosis does not occur in archaea or bacteria, which reproduce via asexual processes such as binary fission.

During meiosis, the genome of a diploid germ cell, which is composed of long segments of DNA packaged into chromosomes, undergoes DNA replication followed by two rounds of division, resulting in four haploid cells. Each of these cells contains one complete set of chromosomes, or half of the genetic content of the original cell. If meiosis produces gametes, these cells must fuse during fertilization to create a new diploid cell, or zygote before any new growth can occur. Thus, the division mechanism of meiosis is a reciprocal process to the joining of two genomes that occurs at fertilization. Because the chromosomes of each parent undergo homologous recombination during meiosis, each gamete, and thus each zygote, will have a unique genetic blueprint encoded in its DNA. Together, meiosis and fertilization constitute sexuality in the eukaryotes, and generate genetically distinct individuals in populations.

Meiosis uses many of the same biochemical mechanisms employed during mitosis to accomplish the redistribution of chromosomes. There are several features unique to meiosis, most importantly the pairing and recombination between homologous chromosomes.

Process

Because meiosis is a "one-way" process, it cannot be said to engage in a cell cycle as mitosis does. However, the preparatory steps that lead up to meiosis are identical in pattern and name to the interphase of the mitotic cell cycle.

Interphase is divided into three phases:

• Growth 1 (G₁) phase: This is a very active period, where the cell synthesizes its vast array of proteins, including the enzymes and structural proteins it will need for growth. In G₁ stage each of the chromosomes consists of a single (very long) molecule of DNA. In humans, at this point cells are 46 chromosomes, 2N, identical to somatic cells.
• Synthesis (S) phase: The genetic material is replicated: each of its chromosomes duplicates, producing 46 chromosomes each made up of two sister chromatids. The cell is still considered diploid because it still contains the same number of centromeres. The identical sister chromatids have not yet condensed into the densely packaged chromosomes visible with the light microscope. This will take place during prophase I in meiosis.
• Growth 2 (G₂) phase: G₂ phase is absent in Meiosis

Interphase is followed by meiosis I and then meiosis II. Meiosis I consists of separating the pairs of homologous chromosome, each made up of two sister chromatids, into two cells. One entire haploid content of chromosomes is contained in each of the resulting daughter cells; the first meiotic division therefore reduces the ploidy of the original cell by a factor of 2.

Meiosis II consists of decoupling each chromosome's sister strands (chromatids), and segregating the individual chromatids into haploid daughter cells. The two cells resulting from meiosis I divide during meiosis II, creating 4 haploid daughter cells. Meiosis I and II are each divided into prophase, metaphase, anaphase, and telophase stages, similar in purpose to their analogous subphases in the mitotic cell cycle. Therefore, meiosis includes the stages of meiosis I (prophase I, metaphase I, anaphase I, telophase I), and meiosis II (prophase II, metaphase II, anaphase II, telophase II).

Meiosis generates genetic diversity in two ways: (1) independent alignment and subsequent separation of homologous chromosome pairs during the first meiotic division allows a random and independent selection of each chromosome segregates into each gamete; and (2) physical exchange of homologous chromosomal regions by homologous recombination during prophase I results in new combinations of DNA within chromosomes.

Phases of Meiosis:

Meiosis I

Meiosis I separates homologous chromosomes, producing two haploid cells (23 chromosomes, N in humans), so meiosis I is referred to as a reductional division. A regular diploid human cell contains 46 chromosomes and is considered 2N because it contains 23 pairs of homologous chromosomes. However, after meiosis I, although the cell contains 46 chromatids it is only considered as being N, with 23 chromosomes, because later in anaphase I the sister chromatids will remain together as the spindle pulls the pair toward the pole of the new cell. In meiosis II, an equational division similar to mitosis will occur whereby the sister chromatids are finally split, creating a total of 4 haploid cells (23 chromosomes, N) per daughter cell from the first division.

Prophase I

During prophase I, DNA is exchanged between homologous chromosomes in a process called homologous recombination. This often results in chromosomal crossover. The new combinations of DNA created during crossover are a significant source of genetic variation, and may result in beneficial new combinations of alleles. The paired and replicated chromosomes are called bivalents or tetrads, which have two chromosomes and four chromatids, with one chromosome coming from each parent. At this stage, non-sister chromatids may cross-over at points called chiasmata (plural; singular chiasma).

                      

Leptotene

The first stage of prophase I is the leptotene stage, also known as leptonema, from Greek words meaning "thin threads".^[1] During this stage, individual chromosomes begin to condense into long strands within the nucleus. However the two sister chromatids are still so tightly bound that they are indistinguishable from one another.

Zygotene

The zygotene stage, also known as zygonema, from Greek words meaning "paired threads",^[1] occurs as the chromosomes approximately line up with each other into homologous chromosomes. This is called the bouquet stage because of the way the telomeres cluster at one end of the nucleus. At this stage, the synapsis (pairing/coming together) of homologous chromosomes takes place.

Pachytene

The pachytene stage, also known as pachynema, from Greek words meaning "thick threads",^[1] contains the following chromosomal crossover. Nonsister chromatids of homologous chromosomes randomly exchange segments of genetic information over regions of homology. Sex chromosomes, however, are not wholly identical, and only exchange information over a small region of homology. Exchange takes place at sites where recombination nodules (the chiasmata) have formed. The exchange of information between the non-sister chromatids results in a recombination of information; each chromosome has the complete set of information it had before, and there are no gaps formed as a result of the process. Because the chromosomes cannot be distinguished in the synaptonemal complex, the actual act of crossing over is not perceivable through the microscope.

Diplotene

During the diplotene stage, also known as diplonema, from Greek words meaning "two threads",^[1] the synaptonemal complex degrades and homologous chromosomes separate from one another a little. The chromosomes themselves uncoil a bit, allowing some transcription of DNA. However, the homologous chromosomes of each bivalent remain tightly bound at chiasmata, the regions where crossing-over occurred. The chiasmata remain on the chromosomes until they are severed in Anaphase I.

In human fetal oogenesis all developing oocytes develop to this stage and stop before birth. This suspended state is referred to as the dictyotene stage and remains so until puberty. In males, only spermatogonia (spermatogenesis) exist until meiosis begins at puberty.

Diakinesis

Chromosomes condense further during the diakinesis stage, from Greek words meaning "moving through". This is the first point in meiosis where the four parts of the tetrads are actually visible. Sites of crossing over entangle together, effectively overlapping, making chiasmata clearly visible. Other than this observation, the rest of the stage closely resembles prometaphase of mitosis; the nucleoli disappear, the nuclear membrane disintegrates into vesicles, and the meiotic spindle begins to form.

Metaphase I

Homologous pairs move together along the metaphase plate: As kinetochore microtubules from both centrioles attach to their respective kinetochores, the homologous chromosomes align along an equatorial plane that bisects the spindle, due to continuous counterbalancing forces exerted on the bivalents by the microtubules emanating from the two kinetochores of homologous chromosomes. The physical basis of the independent assortment of chromosomes is the random orientation of each bivalent along the metaphase plate, with respect to the orientation of the other bivalents along the same equatorial line.

               

Anaphase I

Kinetochore microtubules shorten, severing the recombination nodules and pulling homologous chromosomes apart. Since each chromosome has only one functional unit of a pair of kinetochores, whole chromosomes are pulled toward opposing poles, forming two haploid sets. Each chromosome still contains a pair of sister chromatids. Nonkinetochore microtubules lengthen, pushing the centrioles farther apart. The cell elongates in preparation for division down the center.

Telophase I

The last meiotic division effectively ends when the chromosomes arrive at the poles. Each daughter cell now has half the number of chromosomes but each chromosome consists of a pair of chromatids. The microtubules that make up the spindle network disappear, and a new nuclear membrane surrounds each haploid set. The chromosomes uncoil back into chromatin. Cytokinesis, the pinching of the cell membrane in animal cells or the formation of the cell wall in plant cells, occurs, completing the creation of two daughter cells. Sister chromatids remain attached during telophase I.

Cells may enter a period of rest known as interkinesis or interphase II. No DNA replication occurs during this stage.

Meiosis II

Meiosis II is the second part of the meiotic process. Much of the process is similar to mitosis. The end result is production of four haploid cells (23 chromosomes, 1N in humans) from the two haploid cells (23 chromosomes, 1N * each of the chromosomes consisting of two sister chromatids) produced in meiosis I. The four main steps of Meiosis II are: Prophase II, Metaphase II, Anaphase II, and Telophase II.

Prophase II takes an inversely proportional time compared to telophase I. In this prophase we see the disappearance of the nucleoli and the nuclear envelope again as well as the shortening and thickening of the chromatids. Centrioles move to the polar regions and arrange spindle fibers for the second meiotic division.

In metaphase II, the centromeres contain two kinetochores that attach to spindle fibers from the centrosomes (centrioles) at each pole. The new equatorial metaphase plate is rotated by 90 degrees when compared to meiosis I, perpendicular to the previous plate.

This is followed by anaphase II, where the centromeres are cleaved, allowing microtubules attached to the kinetochores to pull the sister chromatids apart. The sister chromatids by convention are now called sister chromosomes as they move toward opposing poles.

The process ends with telophase II, which is similar to telophase I, and is marked by uncoiling and lengthening of the chromosomes and the disappearance of the spindle. Nuclear envelopes reform and cleavage or cell wall formation eventually produces a total of four daughter cells, each with a haploid set of chromosomes. Meiosis is now complete and ends up with four new daughter cells.

             

Significance

               Meiosis facilitates stable sexual reproduction. Without the halving of ploidy, or chromosome count, fertilization would result in zygotes that have twice the number of chromosomes as the zygotes from the previous generation. Successive generations would have an exponential increase in chromosome count. In organisms that are normally diploid, polyploidy, the state of having three or more sets of chromosomes, results in extreme developmental abnormalities or lethality. Polyploidy is poorly tolerated in most animal species. Plants, however, regularly produce fertile, viable polyploidy. Polyploidy has been implicated as an important mechanism in plant speciation.

              Most importantly, recombination and independent assortment of homologous chromosomes allow for a greater diversity of genotypes in the population. This produces genetic variation in gametes that promote genetic and phenotypic variation in a population of offspring.

********************* 

Unit – II

endoplasmic reticulum:

The endoplasmic reticulum (ER) is an eukaryotic organelle that forms an interconnected network of tubules, vesicles, and cisternae within cells. The lacey membranes of the endoplasmic reticulum were first seen by Keith R. Porter, Albert Claude, and Ernest F. Fullam in 1945.

Structure

                    

The general structure of the endoplasmic reticulum is an extensive membrane network of cisternae (sac-like structures) held together by the cytoskeleton. The phospholipid membrane encloses a space, the cisternal space (or lumen), from the cytosol, which is continuous with the perinuclear space. The functions of the endoplasmic reticulum vary greatly depending on the exact type of endoplasmic reticulum and the type of cell in which it resides. The three varieties are called rough endoplasmic reticulum, smooth endoplasmic reticulum and sarcoplasmic reticulum.

The quantity of RER and SER in a cell can quickly interchange from one type to the other, depending on changing metabolic needs: one type will undergo numerous changes including new proteins embedded in the membranes in order to transform. Also, massive changes in the protein content can occur without any noticeable structural changes, depending on the enzymatic needs of the cell. 

Rough endoplasmic reticulum

The surface of the rough endoplasmic reticulum (RER) is studded with protein-manufacturing ribosomes giving it a "rough" appearance (hence its name).^[2] However, the ribosomes bound to the RER at any one time are not a stable part of this organelle's structure as ribosomes are constantly being bound and released from the membrane. A ribosome only binds to the ER once it begins to synthesize a protein destined for the secretory pathway. Here, a ribosome in the cytosol begins synthesizing a protein until a signal recognition particle recognizes the pre-piece of 5-15 hydrophobic amino acids preceded by a positively charged amino acid. This signal sequence allows the recognition particle to bind to the ribosome, causing the ribosome to bind to the RER and pass the new protein through the ER membrane. The pre-piece is then cleaved off within the lumen of the ER and the ribosome released back into the cytosol.

The membrane of the RER is continuous with the outer layer of the nuclear envelope. Although there is no continuous membrane between the RER and the Golgi apparatus, membrane-bound vesicles shuttle proteins between these two compartments.^[4] Vesicles are surrounded by coating proteins called COPI and COPII. COPII targets vesicles to the golgi and COPI marks them to be brought back to the RER. The RER works in concert with the Golgi complex to target new proteins to their proper destinations. A second method of transport out of the ER are areas called membrane contact sites, where the membranes of the ER and other organelles are held closely together, allowing the transfer of lipids and other small molecules.^[5][6]

The RER is key in multiple functions:

• lysosomal enzymes with a mannose-6-phosphate marker added in the cis-Golgi network
• Secreted proteins, either secreted constitutively with no tag, or regulated secretion involving clathrin and paired basic amino acids in the signal peptide.
• integral membrane proteins that stay imbedded in the membrane as vesicles exit and bind to new membranes. Rab proteins are key in targeting the membrane, SNAP and SNARE proteins are key in the fusion event.
• initial glycosylation as assembly continues. This is either N-linked (O-linking occur in the golgi).

o    N-linked glycosylation: if the protein is properly folded, glycosyltransferase recognizes the AA sequence NXS or NXT (with the S/T residue phosphorylated) and adds a 14 sugar backbone (2 N-acetylglucosamine, 9 branching mannose, and 3 glucose at the end) to the side chain nitrogen of Asn.

Smooth endoplasmic reticulum

The smooth endoplasmic reticulum (SER) has functions in several metabolic processes, including synthesis of lipids and steroids, metabolism of carbohydrates, regulation of calcium concentration, drug detoxification, attachment of receptors on cell membrane proteins, and steroid metabolism. It is connected to the nuclear envelope. Smooth endoplasmic reticulum is found in a variety of cell types (both animal and plant) and it serves different functions in each. The Smooth ER also contains the enzyme glucose-6-phosphatase which converts glucose-6-phosphate to glucose, a step in gluconeogenesis. The SER consists of tubules and vesicles that branch forming a network. In some cells there are dilated areas like the sacs of RER. The network of SER allows increased surface area for the action or storage of key enzymes and the products of these enzymes.

Functions

The endoplasmic reticulum serves many general functions, including the facilitation of protein folding and the transport of synthesized proteins in sacs called cisternae.

Correct folding of newly-made proteins is made possible by several endoplasmic reticulum chaperone proteins, including protein disulfide isomerase (PDI), ERp29, the Hsp70 family member Grp78, calnexin, calreticulin, and the peptidylpropyl isomerase family. Only properly-folded proteins are transported from the rough ER to the Golgi complex.

Transport of proteins

Secretory proteins, mostly glycoproteins, are moved across the endoplasmic reticulum membrane. Proteins that are transported by the endoplasmic reticulum and from there throughout the cell are marked with an address tag called a signal sequence. The N-terminus (one end) of a polypeptide chain (i.e., a protein) contains a few amino acids that work as an address tag, which are removed when the polypeptide reaches its destination. Proteins that are destined for places outside the endoplasmic reticulum are packed into transport vesicles and moved along the cytoskeleton toward their destination.

The endoplasmic reticulum is also part of a protein sorting pathway. It is, in essence, the transportation system of the eukaryotic cell. The majority of endoplasmic reticulum resident proteins are retained in the endoplasmic reticulum through a retention motif. This motif is composed of four amino acids at the end of the protein sequence. The most common retention sequence is KDEL (lys-asp-glu-leu). However, variation on KDEL does occur and other sequences can also give rise to endoplasmic reticulum retention. It is not known if such variation can lead to sub-endoplasmic reticulum localizations. There are three KDEL receptors in mammalian cells, and they have a very high degree of sequence identity. The functional differences between these receptors remain to be established.

Other functions

• Insertion of proteins into the endoplasmic reticulum membrane: Integral membrane proteins are inserted into the endoplasmic reticulum membrane as they are being synthesized (co-translational translocation). Insertion into the endoplasmic reticulum membrane requires the correct topogenic signal sequences in the protein.
• Glycosylation: Glycosylation involves the attachment of oligosaccharides.
• Disulfide bond formation and rearrangement: Disulfide bonds stabilize the tertiary and quaternary structure of many proteins.
• Drug Metabolism: The smooth ER is the site at which some drugs are modified by microsomal enzymes which include the cytochrome P450 enzymes.

Golgi apparatus:

The Golgi apparatus is an organelle found in most eukaryotic cells. It was identified in 1898 by the Italian physician Camillo Golgi and was named after him.

The primary function of the Golgi apparatus is to process and package macromolecules, such as proteins and lipids, after their synthesis and before they make their way to their destination; it is particularly important in the processing of proteins for secretion. The Golgi apparatus forms a part of the cellular endomembrane system.

Structure

The Golgi is composed of membrane-bound stacks known as cisternae (singular: cisterna). Between four and eight are usually present; however, in some protists as many as sixty have been observed. Each cisterna comprises a flattened membrane disk, and carries Golgi enzymes to help or to modify cargo proteins that travel through them. They are found in both plant and animal cells.

The cisternae stack has four functional regions: the cis-Golgi network, medial-Golgi, endo-Golgi, and trans-Golgi network. Vesicles from the endoplasmic reticulum (via the vesicular-tubular clusters) fuse with the cis-Golgi network and subsequently progress through the stack to the trans Golgi network, where they are packaged and sent to the required destination. Each region contains different enzymes which selectively modify the contents depending on where they reside. The cisternae also carry structural proteins important for their maintenance as flattened membranes which stack upon each other.

The trans face of the trans-Golgi network is the face from which vesicles leave the Golgi. These vesicles then proceed to later compartments such as the cell membrane, secretory vesicles or late endosomes.

New cisternae form at the cis-Golgi network. The cis- and trans-Golgi networks are thought to be specialised cisternae leading in and out of the Golgi apparatus.

Function

Cells synthesize a large number of different macromolecules. The Golgi apparatus is integral in modifying, sorting, and packaging these macromolecules for cell secretion (exocytosis) or use within the cell. It primarily modifies proteins delivered from the rough endoplasmic reticulum but is also involved in the transport of lipids around the cell, and the creation of lysosomes. In this respect it can be thought of as similar to a post office; it packages and labels items which it then sends to different parts of the cell.

Enzymes within the cisternae are able to modify substances by the addition of carbohydrates (glycosylation) and phosphates (phosphorylation). In order to do so, the Golgi imports substances such as nucleotide sugars from the cytosol. These modifications may also form a signal sequence which determines their final destination. For example, the Golgi apparatus adds a mannose-6-phosphate label to proteins destined for lysosomes.

The Golgi plays an important role in the synthesis of proteoglycans, which are molecules present in the extracellular matrix of animals. It is also a major site of carbohydrate synthesis. This includes the productions of glycosaminoglycans (GAGs), long unbranched polysaccharides which the Golgi then attaches to a protein synthesised in the endoplasmic reticulum to form proteoglycans.^[4] Enzymes in the Golgi polymerize several of these GAGs via a xylose link onto the core protein. Another task of the Golgi involves the sulfation of certain molecules passing through its lumen via sulphotranferases that gain their sulphur molecule from a donor called PAPs. This process occurs on the GAGs of proteoglycans as well as on the core protein. The level of sulfation is very important to the proteoglycans' signalling abilities as well as giving the proteoglycan its overall negative charge.

The phosphorylation of molecules requires that ATP is imported into the lumen of the Golgi^[5] and then utilised by resident kinases such as casein kinase 1 and casein kinase 2. One molecule that is phosphorylated in the Golgi is Apolipoprotein, which forms a molecule known as VLDL that is a constitute of blood serum. It is thought that the phosphorylation of these molecules is important to help aid in their sorting for secretion into the blood serum.

Vesicular transport

The vesicles that leave the rough endoplasmic reticulum are transported to the cis face of the Golgi apparatus, where they fuse with the Golgi membrane and empty their contents into the lumen. Once inside they are modified, sorted and shipped towards their final destination. As such, the Golgi apparatus tends to be more prominent and numerous in cells synthesising and secreting many substances: plasma B cells, the antibody-secreting cells of the immune system, have prominent Golgi complexes.

Those proteins destined for areas of the cell other than either the endoplasmic reticulum or Golgi apparatus are moved towards the trans face, to a complex network of membranes and associated vesicles known as the trans-Golgi network (TGN). This area of the Golgi is the point at which proteins are sorted and shipped to their intended destinations by their placement into one of at least three different types of vesicles, depending upon the molecular marker they carry:

Type	Description	Example
Exocytotic vesicles (continuous)	Vesicle contains proteins destined for extracellular release. After packaging the vesicles bud off and immediately move towards the plasma membrane, where they fuse and release the contents into the extracellular space in a process known as constitutive secretion.	Antibody release by activated plasma B cells
Secretory vesicles (regulated)	Vesicle contains proteins destined for extracellular release. After packaging the vesicles bud off and are stored in the cell until a signal is given for their release. When the appropriate signal is received they move towards the membrane and fuse to release their contents. This process is known as regulated secretion.	Neurotransmitter release from neurons
Lysosomal vesicles	Vesicle contains proteins destined for the lysosome, an organelle of degradation containing many acid hydrolases, or to lysosome-like storage organelles. These proteins include both digestive enzymes and membrane proteins. The vesicle first fuses with the late endosome, and the contents are then transferred to the lysosome via unknown mechanisms.	Digestive proteases destined for the lysosome

Transport mechanism

The transport mechanism which proteins use to progress through the Golgi apparatus is not yet clear; however a number of hypotheses currently exist. Until recently, the vesicular transport mechanism was favoured but now more evidence is coming to light to support cisternal maturation. The two proposed models may actually work in conjunction with each other, rather than being mutually exclusive. This is sometimes referred to as the combined model.^[3]

• Cisternal maturation model: the cisternae of the Golgi apparatus move by being built at the cis face and destroyed at the trans face. Vesicles from the endoplasmic reticulum fuse with each other to form a cisterna at the cis face, consequently this cisterna would appear to move through the Golgi stack when a new cisterna is formed at the cis face. This model is supported by the fact that structures larger than the transport vesicles, such as collagen rods, were observed microscopically to progress through the Golgi apparatus. This was initially a popular hypothesis, but lost favour in the 1980s. Recently it has made a comeback, as laboratories at the University of Chicago and the University of Tokyo have been able to use new technology to directly observe Golgi compartments maturing.^[7] Additional evidence comes from the fact that COPI vesicles move in the retrograde direction, transporting Endoplasmic Reticulum proteins back to where they belong by recognizing a signal peptide.

• Vesicular transport model: Vesicular transport views the Golgi as a very stable organelle, divided into compartments in the cis to trans direction. Membrane bound carriers transport material between the ER and the different compartments of the Golgi. Experimental evidence includes the abundance of small vesicles (known technically as shuttle vesicles) in proximity to the Golgi apparatus. To direct the vesicles, actin filaments connect packaging proteins to the membrane to ensure that they fuse with the correct compartment.

mitochondria:

In cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 10 micrometers (μm) in diameter. Mitochondria are sometimes described as "cellular power plants" because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction, and may play a role in the aging process. The word mitochondrion comes from the Greek μίτος or mitos, thread + χονδρίον or chondrion, granule.

Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria; whereas in Murinae (rats), 940 proteins encoded by distinct genes have been reported. The mitochondrial proteome is thought to be dynamically regulated. Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

Structure

A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins. The two membranes, however, have different properties. Because of this double-membraned organization, there are five distinct compartments within the mitochondrion. There is the outer mitochondrial membrane, the intermembrane space (the space between the outer and inner membranes), the inner mitochondrial membrane, the crista space (formed by infoldings of the inner membrane), and the matrix (space within the inner membrane).

Outer membrane

The outer mitochondrial membrane, which encloses the entire organelle, has a protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane (about 1:1 by weight). It contains large numbers of integral proteins called porins. These porins form channels that allow molecules 5000 Daltons or less in molecular weight to freely diffuse from one side of the membrane to the other. Larger proteins can enter the mitochondrion if a signaling sequence at their N-terminus binds to a large multisubunit protein called translocase of the outer membrane, which then actively moves them across the membrane. Disruption of the outer membrane permits proteins in the intermembrane space to leak into the cytosol, leading to certain cell death. The mitochondrial outer membrane can associate with the ER membrane, in a structure called MAM (mitochondria-associated ER-membrane). This is important in ER-mitochondria calcium signaling and involved in the transfer of lipids between the ER and mitochondria.

Intermembrane space

The intermembrane space is the space between the outer membrane and the inner membrane. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules such as ions and sugars in the intermembrane space is the same as the cytosol. However, as large proteins must have a specific signaling sequence to be transported across the outer membrane, the protein composition of this space is different than the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c.

Inner membrane

The inner mitochondrial membrane contains proteins with five types of functions:^[6]

1. Those that perform the redox reactions of oxidative phosphorylation
2. ATP synthase, which generates ATP in the matrix
3. Specific transport proteins that regulate metabolite passage into and out of the matrix
4. Protein import machinery.
5. Mitochondria fusion and fission protein

It contains more than 100 different polypeptides, and has a very high protein-to-phospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in beef hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin contains four fatty acids rather than two and may help to make the inner membrane impermeable. Unlike the outer membrane, the inner membrane does not contain porins and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner membrane formed by the action of the enzymes of the electron transport chain.

Cross-sectional image of cristae in rat liver mitochondrion to demonstrate the likely 3D structure and relationship to the inner membrane.

Main article: Crista

The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. For typical liver mitochondria the area of the inner membrane is about five times greater than the outer membrane. This ratio is variable and mitochondria from cells that have a greater demand for ATP, such as muscle cells, contain even more cristae. These folds are studded with small round bodies known as F₁ particles or oxysomes. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function.

Matrix

The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in the production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly-concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle.

Mitochondria have their own genetic material, and the machinery to manufacture their own RNAs and proteins. A published human mitochondrial DNA sequence revealed 16,569 base pairs encoding 37 total genes: 22 tRNA, 2 rRNA, and 13 peptide genes. The 13 mitochondrial peptides in humans are integrated into the inner mitochondrial membrane, along with proteins encoded by genes that reside in the host cell's nucleus.

Organization and distribution

Mitochondria are found in nearly all eukaryotes. They vary in number and location according to cell type. A single highly branched mitochondrion was described in the unicellular alga "Polytomella agilis". Substantial numbers of mitochondria are in the liver, with about 1000–2000 mitochondria per cell making up 1/5th of the cell volume. The mitochondria can be found nestled between myofibrils of muscle or wrapped around the sperm flagellum. Often they form a complex 3D branching network inside the cell with the cytoskeleton. The association with the cytoskeleton determines mitochondrial shape, which can affect the function as well.^[19] Recent evidence suggests vimentin, one of the components of the cytoskeleton, is critical to the association with the cytoskeleton.

 Function

The most prominent roles of mitochondria are to produce ATP (i.e., phosphorylation of ADP) through respiration, and to regulate cellular metabolism. The central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs Cycle. However, the mitochondrion has many other functions in addition to the production of ATP.

 Energy conversion

A dominant role for the mitochondria is the production of ATP, as reflected by the large number of proteins in the inner membrane for this task. This is done by oxidizing the major products of glucose, pyruvate, and NADH, which are produced in the cytosol. This process of cellular respiration, also known as aerobic respiration, is dependent on the presence of oxygen. When oxygen is limited, the glycolytic products will be metabolized by anaerobic respiration, a process that is independent of the mitochondria.^[7] The production of ATP from glucose has an approximately 13-fold higher yield during aerobic respiration compared to anaerobic respiration.^[21] Recently it has been shown that plant mitochondria can produce a limited amount of ATP without oxygen by using the alternate substrate nitrite.

 Pyruvate: the citric acid cycle

Each pyruvate molecule produced by glycolysis is actively transported across the inner mitochondrial membrane, and into the matrix where it is oxidized and combined with coenzyme A to form CO₂, acetyl-CoA, and NADH.

The acetyl-CoA is the primary substrate to enter the citric acid cycle, also known as the tricarboxylic acid (TCA) cycle or Krebs cycle. The enzymes of the citric acid cycle are located in the mitochondrial matrix, with the exception of succinate dehydrogenase, which is bound to the inner mitochondrial membrane as part of Complex II. The citric acid cycle oxidizes the acetyl-CoA to carbon dioxide, and, in the process, produces reduced cofactors (three molecules of NADH and one molecule of FADH₂) that are a source of electrons for the electron transport chain, and a molecule of GTP (that is readily converted to an ATP).

NADH and FADH₂: the electron transport chain

The redox energy from NADH and FADH₂ is transferred to oxygen (O₂) in several steps via the electron transport chain. These energy-rich molecules are produced within the matrix via the citric acid cycle but are also produced in the cytoplasm by glycolysis. Reducing equivalents from the cytoplasm can be imported via the malate-aspartate shuttle system of antiporter proteins or feed into the electron transport chain using a glycerol phosphate shuttle. Protein complexes in the inner membrane (NADH dehydrogenase, cytochrome c reductase, and cytochrome c oxidase) perform the transfer and the incremental release of energy is used to pump protons (H⁺) into the intermembrane space. This process is efficient, but a small percentage of electrons may prematurely reduce oxygen, forming reactive oxygen species such as superoxide. This can cause oxidative stress in the mitochondria and may contribute to the decline in mitochondrial function associated with the aging process.

As the proton concentration increases in the intermembrane space, a strong electrochemical gradient is established across the inner membrane. The protons can return to the matrix through the ATP synthase complex, and their potential energy is used to synthesize ATP from ADP and inorganic phosphate (P_i). This process is called chemiosmosis, and was first described by Peter Mitchell who was awarded the 1978 Nobel Prize in Chemistry for his work. Later, part of the 1997 Nobel Prize in Chemistry was awarded to Paul D. Boyer and John E. Walker for their clarification of the working mechanism of ATP synthase.

Heat production

Under certain conditions, protons can re-enter the mitochondrial matrix without contributing to ATP synthesis. This process is known as proton leak or mitochondrial uncoupling and is due to the facilitated diffusion of protons into the matrix. The process results in the unharnessed potential energy of the proton electrochemical gradient being released as heat. The process is mediated by a proton channel called thermogenin, or UCP1. Thermogenin is a 33kDa protein first discovered in 1973. Thermogenin is primarily found in brown adipose tissue, or brown fat, and is responsible for non-shivering thermogenesis. Brown adipose tissue is found in mammals, and is at its highest levels in early life and in hibernating animals. In humans, brown adipose tissue is present at birth and decreases with age.

Storage of calcium ions:

Mitochondria (M) within a chondrocyte stained for calcium as shown by electron microscopy.

The concentrations of free calcium in the cell can regulate an array of reactions and is important for signal transduction in the cell. Mitochondria can transiently store calcium, a contributing process for the cell's homeostasis of calcium. In fact, their ability to rapidly take in calcium for later release makes them very good "cytosolic buffers" for calcium.^[31][32][33] The endoplasmic reticulum (ER) is the most significant storage site of calcium, and there is a significant interplay between the mitochondrion and ER with regard to calcium. The calcium is taken up into the matrix by a calcium uniporter on the inner mitochondrial membrane. It is primarily driven by the mitochondrial membrane potential. Release of this calcium back into the cell's interior can occur via a sodium-calcium exchange protein or via "calcium-induced-calcium-release" pathways.^[35] This can initiate calcium spikes or calcium waves with large changes in the membrane potential. These can activate a series of second messenger system proteins that can coordinate processes such as neurotransmitter release in nerve cells and release of hormones in endocrine cells.

Additional functions

Mitochondria play a central role in many other metabolic tasks, such as:

• Regulation of the membrane potential
• Apoptosis-programmed cell death^[36]
• Calcium signaling (including calcium-evoked apoptosis)
• Cellular proliferation regulation
• Regulation of cellular metabolism
• Certain heme synthesis reactions
• Steroid synthesis.

Some mitochondrial functions are performed only in specific types of cells. For example, mitochondria in liver cells contain enzymes that allow them to detoxify ammonia, a waste product of protein metabolism. A mutation in the genes regulating any of these functions can result in mitochondrial diseases.

Lysosomes:

Lysosomes are spherical organelles that contain enzymes (acid hydrolases). They break up food so it is easier to digest. They are found in animal cells, while in yeast and plants the same roles are performed by lytic vacuoles. Lysosomes digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria. The membrane around a lysosome allows the digestive enzymes to work at the 4.5 pH they require. Lysosomes fuse with vacuoles and dispense their enzymes into the vacuoles, digesting their contents. They are created by the addition of hydrolytic enzymes to early endosomes from the Golgi apparatus. The name lysosome derives from the Greek words lysis, which means dissolution or destruction, and soma, which means body. They are frequently nicknamed "suicide-bags" or "suicide-sacs" by cell biologists due to their role in autolysis. Lysosomes were discovered by the Belgian cytologist Christian de Duve in 1949.

The size of lysosomes varies from 0.1–1.2 μm. At pH 4.8, the interior of the lysosomes is acidic compared to the slightly alkaline cytosol (pH 7.2). The lysosome maintains this pH differential by pumping protons (H⁺ ions) from the cytosol across the membrane via proton pumps and chloride ion channels. The lysosomal membrane protects the cytosol, and therefore the rest of the cell, from the degradative enzymes within the lysosome. The cell is additionally protected from any lysosomal acid hydrolases that leak into the cytosol as these enzymes are pH-sensitive and function less well in the alkaline environment of the cytosol.

Enzymes

Some important enzymes found within lysosomes include:

• Lipase, which digests lipids
• Amylase, which digest carbohydrates (e.g., sugars)
• Proteases, which digest proteins
• Nucleases, which digest nucleic acids
• phosphoric acid monoesters.

Lysosomal enzymes are synthesized in the cytosol and the endoplasmic reticulum, where they receive a mannose-6-phosphate tag that targets them for the lysosome^{[citation needed]} . Aberrant lysosomal targeting causes inclusion-cell disease, whereby enzymes do not properly reach the lysosome, resulting in accumulation of waste within these organelles^{[citation needed]} and the there about .1 microns

Functions

Lysosomes are the cells' garbage disposal system. They are used for the digestion of macromolecules from phagocytosis (ingestion of other dying cells or larger extracellular material, like foreign invading microbes), endocytosis (where receptor proteins are recycled from the cell surface), and autophagy (wherein old or unneeded organelles or proteins, or microbes that have invaded the cytoplasm are delivered to the lysosome). Autophagy may also lead to autophagic cell death, a form of programmed self-destruction, or autolysis, of the cell, which means that the cell is digesting itself.

Other functions include digesting foreign bacteria (or other forms of waste) that invade a cell and helping repair damage to the plasma membrane by serving as a membrane patch, sealing the wound. In the past, lysosomes were thought to kill cells that were no longer wanted, such as those in the tails of tadpoles or in the web from the fingers of a 3- to 6-month-old fetus. While lysosomes digest some materials in this process, it is actually accomplished through programmed cell death, called apoptosis.^[3][4]

Clinical relevance

There are a number of lysosomal storage diseases that are caused by the malfunction of the lysosomes or one of their digestive proteins, e.g., Tay-Sachs disease, or Pompe's disease. These are caused by a defective or missing digestive protein, which leads to the accumulation of substrates within the cell, impairing metabolism.

In the broad sense, these can be classified as mucopolysaccharidoses, GM₂ gangliosidoses, lipid storage disorders, glycoproteinoses, mucolipidoses, or leukodystrophies.

**********************

UNIT- III

DNA:

DNA, short for deoxyribonucleic acid (pronounced dee-OX-ee-RYE-bow-new-CLAY-ik AH-sid)), is the molecule that contains the genetic code of all organisms. This includes animals, plants, and bacteria. It is also used by some viruses, which are not living organisms but use DNA to infect organisms. DNA is found in each cell in the organism and tells that cells what proteins to make. A cell's proteins determine its function. DNA is inherited by children from their parents. This is why children share traits with their parents, such as skin, hair and eye color. The DNA in a person is a combination of some of the DNA from each of his or her parents.

Structure of DNA

Chemical structure of DNA. The phosphate groups are yellow, the deoxyribonucleic sugars are orange, and the nitrogen bases are green, purple,pink, and blue. The atoms shown are: P=phosphorus O=oxygen N=nitrogen H=hydrogen

      DNA is shaped like a double helix, which is like a ladder twisted into a spiral. Each "leg" of the "ladder" is a line of nucleotides. A nucleotide is a molecule made up of deoxyribose (a kind of sugar with 5 carbon atoms), a phosphate group (made of phosphorus and oxygen), and a nitrogenous base. DNA is made of four types of nitrogenous base:

• adenine (A)/(a)
• thymine (T)/(t)
• cytosine (C)/(c)
• guanine (G)/(g)

  The "rungs" of the DNA ladder are each made of two bases, one base coming from each "leg". The bases connect in the middle: 'A' pairs with 'T', and 'C' pairs with 'G'. The bases are held together by hydrogen bonds.

     Adenine (A) and thymine (T) can pair up because they make two hydrogen bonds, and cytosine (C) and guanine (G) pair up to make three hydrogen bonds. Although the bases are always in fixed pairs, the pairs can come in any order. This way, DNA can write "codes" out of the "letters" that are the bases. These "codes" contain the message that tells the cell what to do.

DNA – A, B and Z forms: 

In a DNA molecule, the two strands are not parallel, but intertwined with each other.  Each strand looks like a helix.  The two strands form a "double helix" structure,  which was first discovered by James D. Watson and Francis Crick in 1953.  In this structure, also known as the B form, the helix makes a turn every 3.4 nm, and the distance between two neighboring base pairs is 0.34 nm.  Hence, there are about 10 pairs per turn.  The intertwined strands make two grooves of different widths, referred to as the major groove and the minor groove, which may facilitate binding with specific proteins.

Figure : The normal right-handed "double helix" structure of DNA, also known as the B form.
            In a solution with higher salt concentrations or with alcohol added, the DNA structure may change to an A form, which is still right-handed, but every 2.3 nm makes a turn and there are 11 base pairs per turn.

Another DNA structure is called the Z form, because its bases seem to zigzag.  Z DNA is left-handed.  One turn spans 4.6 nm, comprising 12 base pairs.  The DNA molecule with alternating G-C sequences in alcohol or high salt solution tends to have such structure.

Figure:   Comparison between B form and Z form.

B-form

• Most common DNA conformation in vivo
• Narrower, more elongated helix than A.
• Wide major groove easily accessible to proteins
• Narrow minor groove
• Favored conformation at high water concentrations (hydyration of minor groove seems to favor B-form)
• Base pairs nearly perpendicular to helix axis
• Sugar pucker C2'-endo

Note that the major groove (at the top, when you have just clicked the button) is wide and easily accessible.

Now the bases are easier to see. Notice how they are stacked upon each other and are nearly perpendicular to the axis of the double helix. Note also that the backbone forms a smooth, continuous curve.

Zoom in on a few base pairs with this button. Hydrogen bonds between the bases are shown in white. You are looking into the major groove. Each base pair stacks on the next similarly, as shown from this view. A-form DNA also stacks in this way, but compare this with Z-DNA, which behaves much differently. DNA is usually found in the B form under physiological conditions. Sometimes kinks are found in the B helix at transcriptional control regions. These kinks can either be intrinsic to the DNA sequence or caused by transcription factor binding.
 

A-form

• Most RNA and RNA-DNA duplex in this form
• shorter, wider helix than B.
• deep, narrow major groove not easily accessible to proteins
• wide, shallow minor groove accessible to proteins, but lower information content than major groove.
• favored conformation at low water concentrations
• base pairs tilted to helix axis
• Sugar pucker C3'-endo (in RNA 2'-OH inhibits C2'-endo conformation)

Note that the major groove (at the top, when you have just clicked the button) is very deep.

Notice how they are stacked upon each other but not perpendicular to the axis of the double helix. They are also displaced to the side of the axis. The result is a wide, short helix. Note also that the backbone forms a smooth, continuous curve.

Zoom in on a few base pairs with this button Hydrogen bonds between the bases are shown in white. You are looking into the major groove. Each base pair stacks on the next similarly, as shown from this view. B-DNA also stacks in this way, but compare this with Z-DNA, which behaves much differently. Essentially all helical RNA is in A form, but DNA can also be found in A form under certain conditions (particularly in RNA-DNA hybrids). The 2'-OH of ribose (shown in white in this view) favors the C3'-endo sugar pucker necessary for A-form geometry. The O2' stericly disfavors the C2'-endo conformation favored in B-DNA.

Z-form

• Helix has left-handed sense
• Can be formed in vivo, given proper sequence and superhelical tension, but function remains obscure.
• Narrower, more elongated helix than A or B.
• Major "groove" not really groove
• Narrow minor groove
• Conformation favored by high salt concentrations, some base substitutions, but requires alternating purine-pyrimidine sequence.
• N2-amino of G H-bonds to 5' PO: explains slow exchange of proton, need for G purine.
• Base pairs nearly perpendicular to helix axis
• GpC repeat, not single base-pair
• P-P distances: vary for GpC and CpG
• GpC stack: good base overlap
• CpG: less overlap.
• Zigzag backbone due to C sugar conformation compensating for G glycosidic bond conformation
• Conformations:
• G; syn, C2'-endo
• C; anti, C3'-endo

Note that the major groove (at the top, when you have just clicked the button) is so wide that it is not really a groove any more.
Now the bases are easier to see. Notice how they are stacked upon each other and are nearly perpendicular to the axis of the double helix. But notice that the base pairs do not stack upon each other equivalently. The backbone also is not a continuous curve, it "zig-zags" back and forth (hence "Z"-DNA)

Notice how the blue bases stack well on the adjacent blue ones, but not on adjacent red ones, and vice versa. So it is the dinucleotide unit, rather than mononucleotide that is the repeating unit of the structure. This explains the need for alternating purines and pyrimidines to form Z-DNA. You can see the same view without the backbone. Going 5' to 3', there is good stacking within the GpC dinucleotide, but not between them (CpG). A top view also illustrates the stacking arrangement. . You can also see this view without the backbone.

DNA Replication:

Before a cell can divide, it must duplicate all its DNA. In eukaryotes, this occurs during S phase of the cell cycle.

The Biochemical Reactions

• DNA replication begins with the "unzipping" of the parent molecule as the hydrogen bonds between the base pairs are broken.
• Once exposed, the sequence of bases on each of the separated strands serves as a template to guide the insertion of a complementary set of bases on the strand being synthesized.
• The new strands are assembled from deoxynucleoside triphosphates.
• Each incoming nucleotide is covalently linked to the "free" 3' carbon atom on the pentose (figure) as
• the second and third phosphates are removed together as a molecule of pyrophosphate (PPi).
• The nucleotides are assembled in the order that complements the order of bases on the strand serving as the template.
• Thus each C on the template guides the insertion of a G on the new strand, each G a C, and so on.
• When the process is complete, two DNA molecules have been formed identical to each other and to the parent molecule.

The Enzymes

• A portion of the double helix is unwound by a helicase.
• A molecule of a DNA polymerase binds to one strand of the DNA and begins moving along it in the 3' to 5' direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix. In eukaryotes, this molecule is called DNA polymerase delta (δ).
• Because DNA synthesis can only occur 5' to 3', a molecule of a second type of DNA polymerase (epsilon, ε, in eukaryotes) binds to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). Another enzyme, DNA ligase I then stitches these together into the lagging strand.

 

 

DNA Replication is Semi conservative:

The structure of DNA suggested to Watson and Crick the mechanism by which DNA — hence genes — could be copied faithfully. They proposed that when the time came for DNA to be replicated, the two strands of the molecule

• separated from each other but
• remained intact as each served as the template for the synthesis of
• a complementary strand.

When the replication process is complete, two DNA molecules — identical to each other and identical to the original — have been produced.

This mode of replication is described as semi conservative: one-half of each new molecule of DNA is old; one-half new.

While Watson and Crick had suggested that this was the way the DNA would turn out to be replicated, proof of the model came from the experiments of M. S. Meselson and F. W. Stahl.

They grew E. coli is a medium using ammonium ions (NH₄⁺) as the source of nitrogen for DNA (as well as protein) synthesis. ¹⁴N is the common isotope of nitrogen, but they could also use ammonium ions that were enriched for a rare heavy isotope of nitrogen, ¹⁵N.

After growing E. coli for several generations in a medium containing ¹⁵NH₄⁺, they found that the DNA of the cells was heavier than normal because of the ¹⁵N atoms in it.

The difference could be detected by extracting DNA from the E. coli cells and spinning it in an ultracentrifuge. The density of the DNA determines where it accumulates in the tube.

Then they transferred more living cells that had been growing in ¹⁵NH₄⁺ to a medium containing ordinary ammonium ions (¹⁴NH₄⁺) and allowed them to divide just once.

The DNA in this new generation of cells was exactly intermediate in density between that of the previous generation and the normal.

This, in itself, is not surprising. It tells us no more than that half the nitrogen atoms in the new DNA are ¹⁴N and half are ¹⁵N. It tells us nothing about their arrangement in the molecules.

However, when the bacteria were allowed to divide again in normal ammonium ions (¹⁴NH₄⁺), two distinct densities of DNA were formed:

• half the DNA was normal and
• half was intermediate.

As this interpretative figure indicates, their results show that DNA molecules are not degraded and reformed from free nucleotides between cell divisions, but instead, each original strand remains intact as it builds a complementary strand from the nucleotides available to it.

This is called semiconservative replication because each daughter DNA molecule is one-half "old" and one-half "new".

Immortal strands. Note that the "old" strand (the red one in the top half of the figure) is immortal because — barring mutations — it will continue to serve as an unchanging template down through the generations.

E. coli is a bacterium, but semiconservative replication of DNA also occurs in eukaryotes. And because each DNA molecule in a eukaryote is incorporated in one chromosome, the replication of entire chromosomes is semiconservative as well. This also means that the eukaryotic chromosome contains one "immortal strand" of DNA.

Ribosomal RNA:

Ribosomal RNA (rRNA) is a type of non-coding ribonucleic acid (RNA) that is a primary and permanent component of ribosomes, the small, cellular particles that form the site of protein synthesis in all living cells. As non-coding RNA, rRNA itself is not translated into a protein, but it does provide a mechanism for decoding messenger RNA (mRNA) into amino acids and interacting with the transfer RNAs (tRNAs) during translation by providing peptidyl transferase activity.

The formation of proteins by rRNA, mRNA, and tRNA is remarkably complex, involving transcription of the various RNAs from DNA, the movement of RNA within a cell, different types of rRNA, and the process of assembling the amino acids in a precise order. And yet this coordinated activity goes on continually in cells, with a single MRNA making several hundred proteins per hour and many thousands of protein molecules per cell generation. With each mammalian cell having millions of ribosomes, and with the human body having many trillions of cells, it is striking to consider how massive, complex, and intricately coordinated is this process of producing proteins for the human body.

Ribosomal RNA (rRNA) is the central component of the ribosome, the protein manufacturing machinery of all living cells. The function of the rRNA is to provide a mechanism for decoding mRNA into amino acids and to interact with the tRNAs during translation by providing peptidyl transferase activity.The tRNA then brings the necessary amino acids corresponding to the appropriate mRNA codon.

                       

Overview

The protein manufacturing unit of all living cells, the ribosome, is composed of ribosomal RNA and protein. It is at the site of the ribosome that messenger RNA's (mRNA) code for linking amino acids together to form new proteins and where transfer RNAs (tRNA) transfer specific amino acids to the growing polypeptide chain during the translation of the mRNA into a protein. The chemical blueprint for the protein product is provided by the mRNA, derived from the DNA genes.

A ribosome can be thought of as a giant enzyme that builds proteins. Its enzymatic activity derives from the presence of the ribosomal RNA (rRNA), which performs the catalytic processes for the synthesis. Meanwhile, the protein portions of the ribosome support the function of the rRNA. More than half the weight of a ribosome is RNA.

Ribosomes are composed of two subunits, named for how rapidly they sediment when subjected to centrifugation. tRNA is sandwiched between the small and large subunits and the ribosome catalyzes the formation of a peptide bond between the two amino acids that are contained in the tRNA.

A ribosome also has 3 binding sites called A, P, and E.

·       The A site in the ribosome binds to an aminoacyl-tRNA (a tRNA bound to an amino acid)

·       The NH2 group of the aminoacyl-tRNA which contains the new amino acid attacks the carboxyl group of peptidyl-tRNA (contained within the P site), which contains the last amino acid of the growing chain called peptidyl transferase reaction

·       The tRNA that was holding on the last amino acid is moved to the E site, and what used to be the aminoacyl-tRNA is now the peptidyl-tRNA

A single mRNA can be translated simultaneously by multiple ribosomes.

Inside the ribosome

The ribosome is composed of two subunits, named for how rapidly they sediment when subject to centrifugation. mRNA is sandwiched between the small and large subunits and the ribosome catalyzes the formation of a peptide bond between the 2 amino acids that are contained in the rRNA.

The ribosome also has 3 binding sites called A, P, and E.

• The A site in the ribosome binds to an aminoacyl-tRNA (a tRNA bound to an amino acid).
• The amino (NH2) group of the aminoacyl-tRNA, which contains the new amino acid, attacks the ester linkage of peptidyl-tRNA (contained within the P site), which contains the last amino acid of the growing chain, forming a new peptide bond. This reaction is catalyzed by peptidyl transferase.
• The tRNA that was holding on the last amino acid is moved to the E site, and what used to be the aminoacyl-tRNA is now the peptidyl-tRNA.

A single mRNA can be translated simultaneously by multiple ribosomes.

Prokaryotes vs. Eukaryotes

Both prokaryotic and eukaryotic can be broken down into two subunits (the S in 16S represents Svedberg units):

Type	Size	Large subunit	Small subunit
prokaryotic	70S	50S (5S, 23S)	30S (16S)
eukaryotic	80S	60S (5S, 5.8S, 28S)	40S (18S)

Note that the S units of the subunits cannot simply be added because they represent measures of sedimentation rate rather than of mass. The sedimentation rate of each subunit is affected by its shape, as well as by its mass.

Translation

Translation is the net effect of proteins being synthesized by ribosomes, from a copy (mRNA) of the DNA template in the nucleus. One of the components of the ribosome (16S rRNA) base pairs complementary to a sequence upstream of the start codon in mRNA.

Importance of rRNA

Ribosomal RNA characteristics are important in medicine and in evolution.

• rRNA is the target of several clinically relevant antibiotics: chloramphenicol, erythromycin, kasugamycin, micrococcin, paromomycin, ricin, sarcin, spectinomycin, streptomycin, and thiostrepton.

• rRNA is the most conserved (least variable) gene in all cells. For this reason, genes that encode the rRNA (rDNA) are sequenced to identify an organism's taxonomic group, calculate related groups, and estimate rates of species divergence. For this reason many thousands of rRNA sequences are known and stored in specialized databases such as RDP-II and the European SSU database.

Transfer RNA:

History

The existence of tRNA was first hypothesized by Francis Crick, based on the assumption that there must exist an adapter molecule capable of mediating the translation of the RNA alphabet into the protein alphabet. Significant research on structure was conducted in the early 1960s by Alex Rich and Don Caspar, two researchers in Boston, the Jacques Fresco group in Princeton University and a United Kingdom group at King's College London. A later publication reported the primary structure in 1965 by Robert W. Holley. The secondary and tertiary structures were derived from X-ray crystallography studies reported independently in 1974 by American and British research groups headed, respectively, by Alexander Rich and Aaron Klug.

Transfer RNA (abbreviated tRNA) is a small RNA molecule (usually about 74-95 nucleotides) that transfers a specific active amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has a 3' terminal site for amino acid attachment. This covalent linkage is catalyzed by an aminoacyl tRNA synthetase. It also contains a three base region called the anticodon that can base pair to the corresponding three base codon region on mRNA. Each type of tRNA molecule can be attached to only one type of amino acid, but because the genetic code contains multiple codons that specify the same amino acid, tRNA molecules bearing different anticodons may also carry the same amino acid.

Structure

Structure of tRNA. CCA tail in orange, Acceptor stem in purple, D arm in red, Anticodon arm in blue with Anticodon in black, T arm in green.

tRNA has primary structure, secondary structure (usually visualized as the cloverleaf structure), and tertiary structure (all tRNAs have a similar L-shaped 3D structure that allows them to fit into the P and A sites of the ribosome). The "cloverleaf structure" becomes the 3D L-shaped structure through coaxial stacking of the helices which is a common RNA Tertiary Structure motif.

1. The 5'-terminal phosphate group.
2. The acceptor stem is a 7-base pair (bp) stem made by the base pairing of the 5'-terminal nucleotide with the 3'-terminal nucleotide (which contains the CCA 3'-terminal group used to attach the amino acid). The acceptor stem may contain non-Watson-Crick base pairs.
3. The CCA tail is a cytosine-cytosine-adenine sequence at the 3' end of the tRNA molecule. This sequence is important for the recognition of tRNA by enzymes critical in translation. In prokaryotes, the CCA sequence is transcribed. In eukaryotes, the CCA sequence is added during processing and therefore does not appear in the tRNA gene.
4. The D arm is a 4 bp stem ending in a loop that often contains dihydrouridine.
5. The anticodon arm is a 5-bp stem whose loop contains the anticodon. It also contains a Y that stands for a modified purine nucleotide.
6. The T arm is a 5 bp stem containing the sequence TΨC where Ψ is a pseudouridine.
7. Bases that have been modified, especially by methylation, occur in several positions outside the anticodon. The first anticodon base is sometimes modified to inosine (derived from adenine) or pseudouridine (derived from uracil).

Anticodon

An anticodon^[1] is a unit made up of three nucleotides that correspond to the three bases of the codon on the mRNA. Each tRNA contains a specific anticodon triplet sequence that can base-pair to one or more codons for an amino acid. For example, one codon for lysine is AAA; the anticodon of a lysine tRNA might be UUU. Some anticodons can pair with more than one codon due to a phenomenon known as wobble base pairing. Frequently, the first nucleotide of the anticodon is one of two not found on mRNA: inosine and pseudouridine, which can hydrogen bond to more than one base in the corresponding codon position. In the genetic code, it is common for a single amino acid to be specified by all four third-position possibilities, or at least by both Pyrimidines and Purines; for example, the amino acid glycine is coded for by the codon sequences GGU, GGC, GGA, and GGG.

To provide a one-to-one correspondence between tRNA molecules and codons that specify amino acids, 61 types of tRNA molecules would be required per cell. However, many cells contain fewer than 61 types of tRNAs because the wobble base is capable of binding to several, though not necessarily all, of the codons that specify a particular amino acid. A minimum of 31 tRNA are required to translate, unambiguously, all 61 sense codons of the standard genetic code.

Aminoacylation

Aminoacylation is the process of adding an aminoacyl group to a compound. It produces tRNA molecules with their CCA 3' ends covalently linked to an amino acid.

Each tRNA is aminoacylated (or charged) with a specific amino acid by an aminoacyl tRNA synthetase. There is normally a single aminoacyl tRNA synthetase for each amino acid, despite the fact that there can be more than one tRNA, and more than one anticodon, for an amino acid. Recognition of the appropriate tRNA by the synthetases is not mediated solely by the anticodon, and the acceptor stem often plays a prominent role.

Reaction:

1. amino acid + ATP → aminoacyl-AMP + PPi
2. aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP

Sometimes, certain organisms can have one or more aminoacyl tRNA synthetases missing. This leads to mischarging of the tRNA by a chemically related amino acid. The correct amino acid is made by enzymes that modify the mischarged amino acid to the correct one.

For example, Helicobacter pylori has glutaminyl tRNA synthetase missing. Thus, glutamate tRNA synthetase mischarges tRNA-glutamine(tRNA-Gln) with glutamate. An amidotransferase then converts the acid side chain of the glutamate to the amide, forming the correctly charged gln-tRNA-Gln.

Binding to ribosome

The ribosome has three binding sites for tRNA molecules: the A (aminoacyl), P (peptidyl), and E (exit) sites. During translation the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This codon specifies the next amino acid to be added to the growing peptide chain. The A site only works after the first aminoacyl-tRNA has attached to the P site. The P-site codon is occupied by peptidyl-tRNA that is a tRNA with multiple amino acids attached as a long chain. The P site is actually the first to bind to aminoacyl tRNA. This tRNA in the P site carries the chain of amino acids that has already been synthesized. The E site is occupied by the empty tRNA as it is about to exit the ribosome.

tRNA genes

Most tRNA genes are transcribed as precursors which undergo further processing. The 5' sequence is removed by RNase P, whereas the 3' end is removed by the tRNase Z enzyme. A notable exception is in the archaeon Nanoarchaeum equitans which does not possess an RNase P enzyme and has a promoter placed such that transcription starts at the 5' end of the mature tRNA. In some bacteria such as E. coli RNase E is used to process the 3' end of the precursor followed by further shortening by exonucleases.

In order to have a better understanding of the role tRNA synthetases, it is important t understand the basic features of tRNA.

tRNa Structure - Cloverleaf Model

The anticodon triplet in the loop at the bottom is complementary to the mRNA codon and will make base pairs with it.

The acceptor stem at the top of the cloverleaf figure is where the amino acid will be attached at the 3' terminus of the tRNA.  This stem always has the sequence 5'...CCA-OH3'.

The D loop and the TyC loop contain a substantial fraction of invariant positions.

The variable loop is variable both in nucleotide composition and in length. 

Messenger RNA:

The "life cycle" of an mRNA in a eukaryotic cell. RNA is transcribed in the nucleus; processed, it is transported to the cytoplasm and translated by the ribosome. At the end of its life, the mRNA is degraded.

Messenger ribonucleic acid (mRNA) is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein. In mRNA as in DNA, genetic information is encoded in the sequence of nucleotides arranged into codons consisting of three bases each. Each codon encodes for a specific amino acid, except the stop codons that terminate protein synthesis. This process requires two other types of RNA: transfer RNA (tRNA) mediates recognition of the codon and provides the corresponding amino acid, while ribosomal RNA (rRNA) is the central component of the ribosome's protein manufacturing machinery.

Structure

The structure of a mature eukaryotic mRNA. A fully processed mRNA includes a 5' cap, 5' UTR, coding region, 3' UTR, and poly(A) tail.

5' cap

The 5' cap is a modified guanine nucleotide added to the "front" (5' end) of the pre-mRNA using a 5'-5'-triphosphate linkage. This modification is critical for recognition and proper attachment of mRNA to the ribosome, as well as protection from 5' exonucleases. It may also be important for other essential processes, such as splicing and transport.

Coding regions

Coding regions are composed of codons, which are decoded and translated (in eukaryotes usually into one and in prokaryotes usually into several) proteins by the ribosome. Coding regions begin with the start codon and end with a stop codon. Generally, the start codon is an AUG triplet and the stop codon is UAA, UAG, or UGA. The coding regions tend to be stabilised by internal base pairs, this impedes degradation.^[3][4] In addition to being protein-coding, portions of coding regions may serve as regulatory sequences in the pre-mRNA as exonic splicing enhancers or exonic splicing silencers.

Untranslated regions

Untranslated regions (UTRs) are sections of the mRNA before the start codon and after the stop codon that are not translated, termed the five prime untranslated region (5' UTR) and three prime untranslated region (3' UTR), respectively. These regions are transcribed with the coding region and thus are exonic as they are present in the mature mRNA. Several roles in gene expression have been attributed to the untranslated regions, including mRNA stability, mRNA localization, and translational efficiency. The ability of a UTR to perform these functions depends on the sequence of the UTR and can differ between mRNAs.

The stability of mRNAs may be controlled by the 5' UTR and/or 3' UTR due to varying affinity for RNA degrading enzymes called ribonucleases and for ancillary proteins that can promote or inhibit RNA degradation.

Translational efficiency, including sometimes the complete inhibition of translation, can be controlled by UTRs. Proteins that bind to either the 3' or 5' UTR may affect translation by influencing the ribosome's ability to bind to the mRNA. MicroRNAs bound to the 3' UTR also may affect translational efficiency or mRNA stability.

Cytoplasmic localization of mRNA is thought to be a function of the 3' UTR. Proteins that are needed in a particular region of the cell can actually be translated there; in such a case, the 3' UTR may contain sequences that allow the transcript to be localized to this region for translation.

Some of the elements contained in untranslated regions form a characteristic secondary structure when transcribed into RNA. These structural mRNA elements are involved in regulating the mRNA. Some, such as the SECIS element, are targets for proteins to bind. One class of mRNA element, the riboswitches, directly bind small molecules, changing their fold to modify levels of transcription or translation. In these cases, the mRNA regulates itself.

Poly(A) tail

The 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the 3' end of the pre-mRNA. This tail promotes export from the nucleus and translation, and protects the mRNA from degradation.

*******************

UNIT – IV

Transcription:

Transcription, or RNA synthesis, is the process of creating an equivalent RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA in the presence of the correct enzymes. During transcription, a DNA sequence is read by RNA polymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement.

Transcription is the first step leading to gene expression. The stretch of DNA transcribed into an RNA molecule is called a transcription unit and encodes at least one gene. If the gene transcribed encodes for a protein, the result of transcription is messenger RNA (mRNA), which will then be used to create that protein via the process of translation. Alternatively, the transcribed gene may encode for either ribosomal RNA (rRNA) or transfer RNA (tRNA), other components of the protein-assembly process, or other ribozymes.

A DNA transcription unit encoding for a protein contains not only the sequence that will eventually be directly translated into the protein (the coding sequence) but also regulatory sequences that direct and regulate the synthesis of that protein. The regulatory sequence before (upstream from) the coding sequence is called the five prime untranslated region (5'UTR), and the sequence following (downstream from) the coding sequence is called the three prime untranslated region (3'UTR).

Transcription has some proofreading mechanisms, but they are fewer and less effective than the controls for copying DNA; therefore, transcription has a lower copying fidelity than DNA replication.

As in DNA replication, DNA is read from 3' → 5' during transcription. Meanwhile, the complementary RNA is created from the 5' → 3' direction. Although DNA is arranged as two antiparallel strands in a double helix, only one of the two DNA strands, called the template strand, is used for transcription. This is because RNA is only single-stranded, as opposed to double-stranded DNA. The other DNA strand is called the coding strand, because its sequence is the same as the newly created RNA transcript (except for the substitution of uracil for thymine). The use of only the 3' → 5' strand eliminates the need for the Okazaki fragments seen in DNA replication.

Transcription is divided into 5 stages: pre-initiation, initiation, promoter clearance, elongation and termination.

Pre-initiation

In eukaryotes, RNA polymerase, and therefore the initiation of transcription, requires the presence of a core promoter sequence in the DNA. Promoters are regions of DNA which promote transcription and are found around -10 to -35 base pairs upstream from the start site of transcription. Core promoters are sequences within the promoter which are essential for transcription initiation. RNA polymerase is able to bind to core promoters in the presence of various specific transcription factors.

The most common type of core promoter in eukaryotes is a short DNA sequence known as a TATA box. The TATA box, as a core promoter, is the binding site for a transcription factor known as TATA binding protein (TBP), which is itself a subunit of another transcription factor, called Transcription Factor II D (TFIID). After TFIID binds to the TATA box via the TBP, five more transcription factors and RNA polymerase combine around the TATA box in a series of stages to form a preinitiation complex. One transcription factor, DNA helicase, has helicase activity and so is involved in the separating of opposing strands of double-stranded DNA to provide access to a single-stranded DNA template. However, only a low, or basal, rate of transcription is driven by the preinitiation complex alone. Other proteins known as activators and repressors, along with any associated coactivators or corepressors, are responsible for modulating transcription rate.

The transcription preinitiation in archaea, formerly a domain of prokaryote, is essentially homologous to that of eukaryotes, but is much less complex.^[5] The archaeal preinitiation complex assembles at a TATA-box binding site; however, in archaea, this complex is composed of only RNA polymerase II, TBP, and TFB (the archaeal homologue of eukaryotic transcription factor II B (TFIIB)).^[6][7]

Initiation

Simple diagram of transcription initiation. RNAP = RNA polymerase

In bacteria, a domain of prokaryotes, transcription begins with the binding of RNA polymerase to the promoter in DNA. RNA polymerase is a core enzyme consisting of five subunits: 2 α subunits, 1 β subunit, 1 β' subunit, and 1 ω subunit. At the start of initiation, the core enzyme is associated with a sigma factor (number 70) that aids in finding the appropriate -35 and -10 base pairs downstream of promoter sequences.

Transcription initiation is more complex in eukaryotes. Eukaryotic RNA polymerase does not directly recognize the core promoter sequences. Instead, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription. Only after certain transcription factors are attached to the promoter does the RNA polymerase bind to it. The completed assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription initiation complex. Transcription in the archaea domain is similar to transcription in eukaryotes.

Promoter clearance

After the first bond is synthesized, the RNA polymerase must clear the promoter. During this time there is a tendency to release the RNA transcript and produce truncated transcripts. This is called abortive initiation and is common for both eukaryotes and prokaroytes^[9]. Abortive initiation continues to occur until the σ factor rearranges, resulting in the transcription elongation complex (which gives a 35 bp moving footprint). The σ factor is released before 80 nucleotides of mRNA are synthesized^[10]. Once the transcript reaches approximately 23 nucleotides, it no longer slips and elongation can occur. This, like most of the remainder of transcription, is an energy-dependent process, consuming adenosine triphosphate (ATP).

Promoter clearance coincides with phosphorylation of serine 5 on the carboxy terminal domain of RNA Pol in prokaryotes, which is phosphorylated by TFIIH.

Elongation

One strand of DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy. Although RNA polymerase traverses the template strand from 3' → 5', the coding (non-template) strand and newly-formed RNA can also be used as reference points, so transcription can be described as occurring 5' → 3'. This produces an RNA molecule from 5' → 3', an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone).

Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be rapidly produced from a single copy of a gene.

Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes, this may correspond with short pauses during transcription that allow appropriate RNA editing factors to bind. These pauses may be intrinsic to the RNA polymerase or due to chromatin structure.

Termination

Bacteria use two different strategies for transcription termination. In Rho-independent transcription termination, RNA transcription stops when the newly synthesized RNA molecule forms a G-C rich hairpin loop followed by a run of U's, which makes it detach from the DNA template. In the "Rho-dependent" type of termination, a protein factor called "Rho" destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex.

Transcription termination in eukaryotes is less understood but involves cleavage of the new transcript followed by template-independent addition of As at its new 3' end, in a process called polyadenylation.

Reverse transcription

Scheme of reverse transcription

Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA. HIV has an RNA genome that is duplicated into DNA. The resulting DNA can be merged with the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an RNA template is called reverse transcriptase. In the case of HIV, reverse transcriptase is responsible for synthesizing a complementary DNA strand (cDNA) to the viral RNA genome. An associated enzyme, ribonuclease H, digests the RNA strand, and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure. This cDNA is integrated into the host cell's genome via another enzyme (integrase) causing the host cell to generate viral proteins which reassemble into new viral particles. Subsequently, the host cell undergoes programmed cell death, apoptosis.

Some eukaryotic cells contain an enzyme with reverse transcription activity called telomerase. Telomerase is a reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase carries an RNA template from which it synthesizes DNA repeating sequence, or "junk" DNA. This repeated sequence of DNA is important because every time a linear chromosome is duplicated it is shortened in length. With "junk" DNA at the ends of chromosomes, the shortening eliminates some of the non-essential, repeated sequence rather than the protein-encoding DNA sequence farther away from the chromosome end. Telomerase is often activated in cancer cells to enable cancer cells to duplicate their genomes indefinitely without losing important protein-coding DNA sequence. Activation of telomerase could be part of the process that allows cancer cells to become technically immortal. However, the true in vivo significance of telomerase has still not been empirically proven.

Post-transcriptional modification:

Post-transcriptional modification is a process in cell biology by which, in eukaryotic cells, primary transcript RNA is converted into mature RNA. A notable example is the conversion of precursor messenger RNA into mature messenger RNA (mRNA), which includes splicing and occurs prior to protein synthesis. This process is vital for the correct translation of the genomes of eukaryotes as the human primary RNA transcript that is produced as a result of transcription contains both exons, which are coding sections of the primary RNA transcript and introns, which are the non coding sections of the primary RNA transcript.^[1]

mRNA processing

The pre-mRNA molecule undergoes three main modifications. These modifications are 5' capping, 3' polyadenylation, and RNA splicing, which occur in the cell nucleus before the RNA is translated.^[2]

5' Processing

Capping

Capping of the pre-mRNA involves the addition of 7-methylguanosine (m⁷G) to the 5' end. In order to achieve this, the terminal 5' phosphate requires removal, which is done by the aid of a phosphatase enzyme. The enzyme guanosyl transferase then catalyses the reaction which produces the diphosphate 5' end. The diphosphate 5' prime end then attacks the α phosphorus atom of a GTP molecule in order to add the guanine residue in a 5'5' triphosphate link. The enzyme S-adenosyl methionine then methylates the guanine ring at the N-7 position. This type of cap, with just the (m⁷G) in position is called a cap 0 structure. The ribose of the adjacent nucleotide may also be methylated to give a cap 1. Methylation of nucleotides downstream of the RNA molecule produce cap 2, cap 3 structures and so on. In these cases the methyl groups are added to the 2' OH groups of the ribose sugar. The cap protects the 5' end of the primary RNA transcript from attack by ribonucleases that have specificity to the 3'5' phosphodiester bonds.^[3]

3' Processing

Cleavage and Polyadenylation

The pre-mRNA processing at the 3' end of the RNA molecule involves cleavage of its 3' end and then the addition of about 200 adenine residues to form a poly(A) tail. The cleavage and adenylation reactions occur if a polyadenylation signal sequence (5'- AAUAAA-3') is located near the 3' end of the pre-mRNA molecule, which is followed by another sequence, which is usually (5'-CA-3'). The second signal is the site of cleavage. A GU-rich sequence is also usually present further downstream on the pre-mRNA molecule. After the synthesis of the sequence elements, two multisubunit proteins called cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CStF) are transferred from RNA Polymerase II to the RNA molecule. The two factors bind to the sequence elements. A protein complex forms which contains additional cleavage factors and the enzyme Polyadenylate Polymerase (PAP). This complex cleaves the RNA between the polyadenylation sequence and the GU-rich sequence at the cleavage site marked by the (5'-CA-3') sequences. Poly(A) polymerase then adds about 200 adenine units to the new 3' end of the RNA molecule using ATP as a precursor. As the poly(A) tails is synthesised, it binds multiple copies of poly(A) binding protein, which protects the 3'end from ribonuclease digestion.^[3]

Splicing

RNA splicing is the process by which introns, regions of RNA that do not code for protein, are removed from the pre-mRNA and the remaining exons connected to re-form a single continuous molecule. Although most RNA splicing occurs after the complete synthesis and end-capping of the pre-mRNA, transcripts with many exons can be spliced co-transcriptionally.^[4] The splicing reaction is catalyzed by a large protein complex called the spliceosome assembled from proteins and small nuclear RNA molecules that recognize splice sites in the pre-mRNA sequence. Many pre-mRNAs, including those encoding antibodies, can be spliced in multiple ways to produce different mature mRNAs that encode different protein sequences. This process is known as alternative splicing, and allows production of a large variety of proteins from a limited amount of DNA.

Protein Synthesis:

The Steps in Protein Synthesis

 

 

1. Initiation

• The small subunit of the ribosome binds to a site "upstream" (on the 5' side) of the start of the message.
• It proceeds downstream (5' -> 3') until it encounters the start codon AUG. (The region between the cap and the AUG is known as the 5'-untranslated region [5'-UTR].)
• Here it is joined by the large subunit and a special initiator tRNA.
• The initiator tRNA binds to the P site (shown in pink) on the ribosome.
• In eukaryotes, initiator tRNA carries methionine (Met). (Bacteria use a modified methionine designated fMet.)

2. Elongation

• An aminoacyl-tRNA (a tRNA covalently bound to its amino acid) able to base pair with the next codon on the mRNA arrives at the A site (green) associated with:
• an elongation factor (called EF-Tu in bacteria)
• GTP (the source of the needed energy)
• The preceding amino acid (Met at the start of translation) is covalently linked to the incoming amino acid with a peptide bond (shown in red).
• The initiator tRNA is released from the P site.
• The ribosome moves one codon downstream.
• This shifts the more recently-arrived tRNA, with its attached peptide, to the P site and opens the A site for the arrival of a new aminoacyl-tRNA.
• This last step is promoted by another protein elongation factor (called EF-G in bacteria) and the energy of another molecule of GTP.

Note: the initiator tRNA is the only member of the tRNA family that can bind directly to the P site. The P site is so-named because, with the exception of initiator tRNA, it binds only to a peptidyl-tRNA molecule; that is, a tRNA with the growing peptide attached.

The A site is so-named because it binds only to the incoming aminoacyl-tRNA; that is the tRNA bringing the next amino acid. So, for example, the tRNA that brings Met into the interior of the polypeptide can bind only to the A site.

3. Termination

• The end of translation occurs when the ribosome reaches one or more STOP codons (UAA, UAG, UGA). (The nucleotides from this point to the poly(A) tail make up the 3'-untranslated region [3'-UTR] of the mRNA.)
• There are no tRNA molecules with anticodons for STOP codons.
• However, protein release factors recognize these codons when they arrive at the A site.
• Binding of these proteins —along with a molecule of GTP — releases the polypeptide from the ribosome.
• The ribosome splits into its subunits, which can later be reassembled for another round of protein synthesis.

4.  Polysomes Formation:

A single mRNA molecule usually has many ribosomes traveling along it, in various stages of synthesizing the polypeptide. This complex is called a polysome

 

 

 

 

 

Post translational modification:

Posttranslational modification (PTM) is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis for many proteins.  The bottom of this diagram shows the modification of primary structure of insulin, as described.

A protein (also called a polypeptide) is a chain of amino acids. During protein synthesis, 20 different amino acids can be incorporated in proteins. After translation, the posttranslational modification of amino acids extends the range of functions of the protein by attaching to it other biochemical functional groups such as acetate, phosphate, various lipids and carbohydrates, by changing the chemical nature of an amino acid (e.g. citrullination) or by making structural changes, like the formation of disulfide bridges.

Also, enzymes may remove amino acids from the amino end of the protein, or cut the peptide chain in the middle. For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds. Also, most nascent polypeptides start with the amino acid methionine because the "start" codon on mRNA also codes for this amino acid. This amino acid is usually taken off during post-translational modification.

Other modifications, like phosphorylation, are part of common mechanisms for controlling the behavior of a protein, for instance activating or inactivating an enzyme.

Proteolytic Cleavage

Most proteins undergo proteolytic cleavage following translation. The simplest form of this is the removal of the initiation methionine. Many proteins are synthesized as inactive precursors that are activated under proper physiological conditions by limited proteolysis. Pancreatic enzymes and enzymes involved in clotting are examples of the latter. Inactive precursor proteins that are activated by removal of polypeptides are termed proproteins.

A complex example of post-translational processing of a preproprotein is the cleavage of prepro-opiomelanocortin (POMC) synthesized in the pituitary (see the Peptide Hormones page for discussion of POMC). This preproprotein undergoes complex cleavages, the pathway of which differs depending upon the cellular location of POMC synthesis.

Another is example of a preproprotein is insulin. Since insulin is secreted from the pancreas it has a prepeptide. Following cleavage of the 24 amino acid signal peptide the protein folds into proinsulin. Proinsulin is further cleaved yielding active insulin which is composed of two peptide chains linked togehter through disulfide bonds.

Still other proteins (of the enzyme class) are synthesized as inactive precursors called zymogens. Zymogens are activated by proteolytic cleavage such as is the situation for several proteins of the blood clotting cascade.

Acylation

Many proteins are modified at their N-termini following synthesis. In most cases the initiator methionine is hydrolyzed and an acetyl group is added to the new N-terminal amino acid. Acetyl-CoA is the acetyl donor for these reactions. Some proteins have the 14 carbon myristoyl group added to their N-termini. The donor for this modification is myristoyl-CoA. This latter modification allows association of the modified protein with membranes. The catalytic subunit of cyclicAMP-dependent protein kinase (PKA) is myristoylated.

Methylation

Post-translational methylation of proteins occurs on nitrogens and oxygens. The activated methyl donor is S-adenosylmethionine (SAM). The most common methylations are on the ε-amine of lysine residues.  Additional nitrogen methylations are found on the imidazole ring of histidine, the guanidino moiety of arginine and the R-group amides of glutamate and aspartate. N-methylation is a permanent modification and there are no known mammalian enzymes that can remove the methyl group. Methylation of the oxygen of the R-group carboxylates of lgutamate and aspartate also takes place and forms methyl esters. Proteins can also be methylated on the thiol R-group of cysteine. Methylation of histones in DNA is an important regulator of chromatin structure and consequently of transcriptional activity.

As indicated below, many proteins are modified at their C-terminus by prenylation near a cysteine residue in the consensus CAAX. Following the prenylation reaction the protein is cleaved at the peptide bond of the cysteine and the carboxylate residue is methylated by a prenylated protein methyltransferase. One such protein that undergoes this type of modification is the proto-oncogene RAS.

Phosphorylation

Post-translational phosphorylation is one of the most common protein modifications that occurs in animal cells. The vast majority of phosphorylations occur as a mechanism to regulate the biological activity of a protein and as such are transient. In other words a phosphate (or more than one in many cases) is added and later removed.

Physiologically relevant examples are the phosphorylations that occur in glycogen synthase and glycogen phosphorylase in hepatocytes in response to glucagon release from the pancreas. Phosphorylation of synthase inhibits its activity, whereas, the activity of phosphorylase is increased. These two events lead to increased hepatic glucose delivery to the blood.

The enzymes that phosphorylate proteins are termed kinases and those that remove phosphates are termed phosphatases. Protein kinases catalyze reactions of the following type:

ATP + protein <——> phosphoprotein + ADP

In animal cells serine, threonine and tyrosine are the amino acids subject to phosphorylation. The largest group of kinases are those that phsophorylate either serines or threonines and as such are termed serine/threonine kinases. The ratio of phosphorylation of the three different amino acids is approximately 1000/100/1 for serine/threonine/tyrosine.

Although the level of tyrosine phosphorylation is minor, the importance of phosphorylation of this amino acid is profound. As an example, the activity of numerous growth factor receptors is controlled by tyrosine phosphorylation.

Sulfation

Sulfate modification of proteins occurs at tyrosine residues such as in fibrinogen and in some secreted proteins (eg gastrin). The universal sulfate donor is 3'-phosphoadenosyl-5'-phosphosulphate (PAPS).

Since sulfate is added permanently it is necessary for the biological activity and not used as a regulatory modification like that of tyrosine phosphorylation.

Prenylation

Prenylation refers to the addition of the 15 carbon farnesyl group or the 20 carbon geranylgeranyl group to acceptor proteins, both of which are isoprenoid compounds derived from the cholesterol biosynthetic pathway. The isoprenoid groups are attached to cysteine residues at the carboxy terminus of proteins in a thioether linkage (C-S-C). A common consensus sequence at the C-terminus of prenylated proteins has been identified and is composed of CAAX, where C is cysteine, A is any aliphatic amino acid (except alanine) and X is the C-terminal amino acid. In order for the prenylation reaction to occur the three C-terminal amino acids (AAX) are first removed. Following attachment of the prenyl group the carboxylate of the cysteine is methylated in a reaction utilizing S-adenosylmethionine as the methyl donor.

In addition to numerous prenylated proteins that contain the CAAX consensus, prenylation is known to occur on proteins of the RAB family of RAS-related G-proteins. There are at least 60 proteins in this family that are prenylated at either a CC or CXC element in their C-termini. The RAB family of proteins are involved in signaling pathways that control intracellular membrane trafficking.

Some of the most important proteins whose functions depend upon prenylation are those that modulate immune responses. These include proteins involved in leukocyte motility, activation, and proliferation and endothelial cell immune functions. It is these immune modulatory roles of many prenylated proteins that are the basis for a portion of the anti-inflammatory actions of the statin class of cholesterol synthesis-inhibiting drugs due to a reduction in the synthesis of farnesylpyrophosphate and geranylpyrophosphate and thus reduced extent of inflammatory events. Other important examples of prenylated proteins include the oncogenic GTP-binding and hydrolyzing protein RAS and the γ-subunit of the visual protein transducin, both of which are farnesylated. In addition, numerous GTP-binding and hydrolyzing proteins (termed G-proteins) of signal transduction cascades have γ-subunits modified by geranylgeranylation.

Vitamin C-Dependent Modifications

Modifications of proteins that depend upon vitamin C as a cofactor include proline and lysine hydroxylations and carboxy terminal amidation. The hydroxylating enzymes are identified as prolyl hydroxylase and lysyl hydroxylase. The donor of the amide for C-terminal amidation is glycine. The most important hydroxylated proteins are the collagens. Several peptide hormones such as oxytocin and vasopressin have C-terminal amidation.

Vitamin K-Dependent Modifications

Vitamin K is a cofactor in the carboxylation of glutamic acid residues. The result of this type of reaction is the formation of a γ-carboxyglutamate (gamma-carboxyglutamate), referred to as a gla residue.

Structure of a gla Residue

The formation of gla residues within several proteins of the blood clotting cascade is critical for their normal function. The presence of gla residues allows the protein to chelate calcium ions and thereby render an altered conformation and biological activity to the protein. The coumarin-based anticoagulants, warfarin and dicumarol function by inhibiting the carboxylation reaction.

Selenoproteins

Selenium is a trace element and is found as a component of several prokaryotic and eukaryotic enzymes that are involved in redox reactions. The selenium in these selenoproteins is incorporated as a unique amino acid, selenocysteine, during translation. A particularly important eukaryotic selenoenzyme is glutathione peroxidase. This enzyme is required during the oxidation of glutathione by hydrogen peroxide (H₂O₂) and organic hydroperoxides.

************************

UNIT – V

Spontaneous and Induced Mutations:

In general, the appearance of a new mutation is a rare event. Most mutations that were originally studied occurred spontaneously. This class of mutation is termed spontaneous mutations. Historically, geneticists recognized these in nature. The mutations were collected, and the inheritance of these mutations were analyzed. But these mutations clearly represent only a small number of all possible mutations. These mutations are called induced mutations.

The spontaneous mutation rate varies. Large genes provide a large target and tend to mutate more frequently. A study of the five coat color loci in mice showed that the rate of mutation ranged from 2 x 10^-6 to 40 x 10^-6 mutations per gamete per gene. Data from several studies on eukaryotic organisms shows that in general the spontaneous mutation rate is 2-12 x 10^-6 mutations per gamete per gene. Given that the human genome contains 100,000 genes, we can conclude that we would predict that 1-5 human gametes would contain a mutation in some gene.

Mutations can be induced by several methods. The three general approaches used to generate mutations are radiation, chemical and transposon insertion. The first induced mutations were created by treating Drosophila with X-rays. Using this a pproach Mueller to induce lethal mutations. In addition to X-rays, other types of radiation treatments that have proven useful include gamma rays and fast neutron bombardment. These treatments can induce point mutations (changes in a single nucleotide) or deletions (loss of a chromosomal segment).

Chemical mutagens work mostly by inducing point mutations. Point mutations occur when a single base pair of a gene is changed. These changes are classified as transitions or transversions. Transitions occur when a purine is convert ed to a purine (A to G or G to A) or a pyrimide is converted to a pyrimidine (T to C or C to T). A transversion results when a purine is converted to a pyrimidine or a pyrimidine is converted to a purine. A transversion example is adenine being converte d to a cytosine. You can determine other examples.

Two major classes of chemical mutagens are routinely used. These are alkylating agents and base analogs. Each has a specific effect on DNA. Alkylating agents [such as ethyl methane sulphonate (EMS), ethyl ethane sulphonate (EES) and musta rd gas] can mutate both replicating and non-replicating DNA. By contrast, a base analog (5-bromouracil and 2-aminopurine) only mutate DNA when the analog is incorporated into replicating DNA. Each class of chemical mutagen has specific effects that can lead to transitions, transversions or deletions.

Scientists are now using the power of transposable elements to create new mutations. Transposable elements are mobile pieces of DNA that can move from one location in a geneome to another. Often when they move to a new location, the result is a new mutan t. The mutant arises because the presence of a piece of DNA in a wild type gene disrupts the normal function of that gene. As more and more is being learned about genes and genomes, it is becoming apparent that transposable elements are a power source f or creating insertional mutants.

The detailed knowledge of the structure and funciton of transposable elements is now being applied in the pursuit of new mutations. Stocks are created in which a specific type of element is present. This stock is then crossed to a genetic stock that doe s not contain the element. Once that element enters the virgin stock, it can begin to move around that genome. By carefully observing the offspring, new mutants can be discovered. This approach to developing mutants is termed insertional mutagenesis.

mutagenesis:

Mutations can arise randomly or as a result of specific targeting (site-directed mutagenesis).

Remember that DNA is a chemical molecule and is subject to reaction with other chemicals. These reactions will be affected by HEAT - which will speed up the rate of spontaneous reactions.

The reaction of different chemicals with DNA is both random and nonrandom. The different bases have different chemical reactivities and so any particular chemical agent will react differently with the different bases, however, there will be no discrimination between bases of the same type.

 Spontaneous mutagenesis

Spontaneous mutations arise most likely as a result of errors during DNA replication. They occur at characteristic frequency for any given organism. For E. coli, the rate of spontaneous mutagenesis is approximately 1 in 10^­7.

Mistakes during replication arise due to incorrect base-pairing which is in turn due to:

·         Tautomerization

·         chemical change -- e.g. spontaneous deamination

Mistakes which persist after replication as a result of defective repair systems give rise to mutations.

 Induced mutagenesis

Mutations can occur as a result of treating DNA with a variety of chemicals or other agents.

 Formation of pyrimidine dimers

UV light is particularly effective at generating pyrimidine dimers. The conjugated ring systems of adjacent thymine bases in a polynucleotide chain will absorb UV light and form a cyclobutane ring which links carbons 5 and 6 of each pyrimidine ring to one another. Adjacent thymine and cytidine bases can also be photoactivated to form a 6-4 linkage between the two bases.

These do not usually generate errors unless DNA repair systems are faulty.

Pyrimidine dimers can be recognized by photolyase which binds to the photodimer and, in the presence of visible light, will split the dimer.

All photolyases contain 2 chromophores. One is always FADH₂; the other is either a folate or deazaflavin. All photolyases require light in the range 300-500 nm. The E. coli enzyme requires light in the range 365-400 nm.

Deamination 

·         Adenine is deaminated to hypoxanthine which prefers to pair with cytosine, hence the transition AT -> GC results.

·         Cytosine is deaminated to uracil, hence the transition CG -> TA results.

·         Guanine is also deaminated -- to xanthine -- but since xanthine also pairs with cytosine this has no mutagenic effect.

Common deaminating agents are nitrous acid (HNO₂), hydroxylamine (HONH₂) and bisulfite (HSO₃^­). Of these, only nitrous acid can enter cells and is, therefore, suitable as an in vivo mutagen. Bisulfite is also useful because it only reacts with single-stranded DNA; therefore, it can be used as a probe to determine which regions of a DNA molecule are (temporarily) double-stranded. Hydroxylamine specifically induces GC -> AT transitions.These lesions can be repaired by an N-glycosylase and AP endonuclease.

           Spontaneous deaminations are not found in random locations. It is clear that HOTSPOTS exist at which the frequencies of mutation are much greater than usual. Analysis of hotspots in the lacI gene has shown that the hotspots are due to the presence of 5-methyl-cytosine, rather than cytosine at these positions. 5-Me-C undergoes oxidative deamination much more readily than cytosine and forms thymine. Since thymine is a normal constituent of DNA, there is no lesion to repair - hence the mutational hotspot.

Cytosine is methylated by the Dcm methylase which recognizes the sequence 5'-CCWGG-3'. The enzyme methylates the second cytosine in this sequence.
[Note that W is the symbol used when either A or T can be found.]

 Oxidative damage

Oxidative damage can be caused by superoxide radicals, hydrogen peroxide, or hydroxide radicals. The most important type of oxidative damage is the formation of 8-oxo-guanine which will pair with adenine and generate transversions.

 Alkylation

These agents modify bases and/or phosphates by alkylating them. The DNA becomes distorted as a result and the ability of proteins to recognize and bind correctly is hindered. They are among the most powerful mutagens. Examples are:

MMS - methyl methane sulfonate

EMS - ethyl methane sulfonate (nitrogen mustard gas)

NTG - nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine - MNNG)

nitrosoguanidine is particularly effective because it attacks single-stranded DNA at replication forks.

Ethylnitrosourea

Ethylnitrosourea is often used to probe which phosphates are required for proteins that bind to specific DNA sites.

            These chemicals attack different reactive groups. The most reactive atoms for alkylation are the N⁷ of guanine and the N³ of adenine. Alkylation at these positions will distort the double helix. Such distortions can be repaired by an N-glycosylase and an error-prone repair system. For example, the alkA gene codes for a N-glyosylase which removes 3-methyl-guanine and 3-methyl-adenine.

Alkylation can also occur at oxygen atoms: the O⁶ of guanine and the O⁴ of thymine. This will result in mispairing of base pairs but does not generate major distortions.. O-6-ethylguanine will pair with thymine and O-4-ethylthymine will pair with guanine. Both changes cause transitions. These lesions can be repaired by methyltransferases which remove the methyl group by transferring it to themselves.

The repair of damage due to alkylation is also intimately connected with the adaptive response.

Base analogues

Nucleotide base analogues can be incorporated into DNA during replication which will result in erroneous pairing. Examples are:

5-bromo-uracil

This analogue tautomerizes more readily than thymine. As a result, it will pair with guanine more frequently. The result is a TA -> CG transition.

2-aminopurine

This analogue normally pairs with thymine but when it is protonated, it will pair with cytosine. The result is an AT -> GC transition.

Repair of these lesions uses the DNA Mismatch Repair System.

Frameshift mutations

Frame shift mutations are generally caused by intercalating agents. These chemicals intercalate (insert) themselves between base pairs in a double helix or between ring-stacked bases in a polynucleotide chain thus distorting the structure.

The best known intercalating agents are ethidium bromide and acridine orange.

 The Adaptive Response

E. coli cells that are exposed to low doses of an alkylating agent (such as NTG) are better able to handle subsequent high doses of an alkylating agent. In other words, the cells seem to adapt to be able to handle the agent. This is called the adaptive response.

The low initial dose of mutagen induces the synthesis of four gene products (Ada, AlkA, AlkB, AidB) which are then able to deal with the subsequent high dosage. 

·         Ada is both a methyl transferase and a transcriptional activator. Its function is described below.

·         AlkA is a glycosylase that removes N7-methylguanine, N3-methylpurines and O2-methylpyrimidines. The ABASIC sites generated are then excised by an AP endonuclease and repaired by PolI and DNA ligase.

·         AlkB has an unknown function in repair. alkB^­ mutants are hypersensitive to MMS and DMS and are poor at reactivating MMS treated bacteriophage l.

·         AidB resembles the isovaleryl CoA dehydrogenase and may be involved in metabolizing alkylating agents such as NTG.

Ada

The key molecule in the adaptive response is the Ada protein. It is both a repair enzyme and a regulatory protein. The repair activity stimulates the regulatory function; the regulatory action stimulates furthrt repair.

The Ada protein is a methyltransferase which is expressed at low levels in normal cells. Methylation is irreversible.

Its function is to sense the presence of alkylated DNA. If it encounters O-6-methylguanine or O-4-methylthymine, both of which are highly mutagenic, it catalyzes the transfer of the methyl group to its own Cys321. If it encounters a methylated phosphate group, it transfers the methyl group to its own Cys69. While methylated phosphates are innocuous, they serve as an indicator of abnormal methylation activity which is then conveyed to Ada.

Ada methylated at Cys69 becomes a transcriptional activator which functions at three promoters:

·         the aidB promoter

transcription from this promoter allows expression of the AidB protein.

·         the ada promoter

transcription from this promoter expresses both the ada and alkB genes. Since Ada regulates its own expression, it is autoregulatory.

·         the alkA promoter

transcription from this promoter allows expression of AlkA .

            Activation at the ada and aidB promoters is mechanistically similar.

RNA polymerase binds poorly to the ada and aidB promoters in the absence of Ada. In both cases, the carboxyl terminal domain (CTD) of the RNA polymerase a subunit (a-CTD) contacts the UP promoter element (an A/T rich region located between 40 and 60 bp upstream of the startpoint of transcription). However, binding of ^MeAda is required to formation a ternary complex that is capable of transcription initiation.

The amino-terminal domain (NTD) of ^MeAda binds to its binding site which is located in the UP promoter element (between -57 and -45) in the ada promoter and (between -55 and -43) in the aidB promoter. Binding is independent of RNA polymerase.

The carboxyl terminal domain of ^MeAda then interacts with the s subunit of RNA polymerase. This interaction is mediated by a region of negatively charged residues (E574, E575, E591, E605) in the s⁷⁰ subunit of RNA polymerase. This stabilises the otherwise weak interaction between RNA polymerase and the ada promoter. (Note that E575 and E591 have also been implicated in activation by the PhoB and lcI proteins.)

Some evidence:

o    The Ada NTD will bind to DNA by itself as long as Cys69 is methylated. The affinity and specificity of binding are the same as that of the full-length protein.

o    The Ada NTD cannot activate transcription at either the ada or aidB promoters.

It is believed that methylation at Cys69 converts Ada from an inactive to an active regulatory form as a result of a conformational change. Cys321 is buried in the interior of the protein. However, upon methylation of Cys69, the positively charged region around Cys321 becomes exposed on the surface of the protein. Methylation of Cys321 stabilizes the altered conformation that permits interaction of Ada and s⁷⁰. Methylation of Cys321 per se plays only a small role in activation. While it is not necessary for activation, methylation will result in optimum activation.

Activation at the alkA promoter is mechanistically different.

At this promoter, the Ada binding site is located between -47 and -35. This positions Ada so that it can interact both with the a-CTD of RNA polymerase and with the s⁷⁰ subunit.

In this case it is the amino-terminal domain (NTD) of ^MeAda which is responsible for interacting with RNA polymerase as well as for binding to DNA. The interaction is mediated by positively charged residues (K593, K597, R603) in the s⁷⁰ subunit of RNA polymerase and (presumably) a negatively charged residue in Ada. There is also an interaction between Ada and the a-CTD of RNA polymerase which contributes to the binding of RNA polymerase to the promoter.

Non-methylated Ada is able to activate the alkA promoter weakly. This does not happen at the ada and aidB promoters.

 

DNA Damage and Repair Mechanisms:

Importance

DNA in the living cell is subject to many chemical alterations (a fact often forgotten in the excitement of being able to do DNA sequencing on dried and/or frozen specimens). If the genetic information encoded in the DNA is to remain uncorrupted, any chemical changes must be corrected.

A failure to repair DNA produces a mutation.

The recent publication of the human genome  has already revealed 130 genes whose products participate in DNA repair. More will probably be identified soon.

Agents that Damage DNA

• Certain wavelengths of radiation
• ionizing radiation such as gamma rays and x-rays
• ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B that penetrates the ozone shield [Link].
• Highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways.
• Chemicals in the environment
• many hydrocarbons, including some found in cigarette smoke
• some plant and microbial products, e.g. the aflatoxins produced in moldy peanuts
• Chemicals used in chemotherapy, especially chemotherapy of cancers

Types of DNA Damage

1. All four of the bases in DNA (A, T, C, G) can be covalently modified at various positions.
• One of the most frequent is the loss of an amino group ("deamination") — resulting, for example, in a C being converted to a U.
2. Mismatches of the normal bases because of a failure of proofreading during DNA replication.
• Common example: incorporation of the pyrimidine U (normally found only in RNA) instead of T.
3. Breaks in the backbone.
• Can be limited to one of the two strands (a single-stranded break, SSB) or
• on both strands (a double-stranded break (DSB).
• Ionizing radiation is a frequent cause, but some chemicals produce breaks as well.
4. Crosslinks Covalent linkages can be formed between bases
• on the same DNA strand ("intrastrand") or
• on the opposite strand ("interstrand").

Several chemotherapeutic drugs used against cancers crosslink DNA.

Repairing Damaged Bases

Damaged or inappropriate bases can be repaired by several mechanisms:

• Direct chemical reversal of the damage
• Excision Repair, in which the damaged base or bases are removed and then replaced with the correct ones in a localized burst of DNA synthesis. There are three modes of excision repair, each of which employs specialized sets of enzymes.
1. Base Excision Repair (BER)
2. Nucleotide Excision Repair (NER)
3. Mismatch Repair (MMR)

Direct Reversal of Base Damage

Perhaps the most frequent cause of point mutations in humans is the spontaneous addition of a methyl group (CH₃-) (an example of alkylation) to Cs followed by deamination to a T. Fortunately, most of these changes are repaired by enzymes, called glycosylases, that remove the mismatched T restoring the correct C. This is done without the need to break the DNA backbone (in contrast to the mechanisms of excision repair described below).

Some of the drugs used in cancer chemotherapy ("chemo") also damage DNA by alkylation. Some of the methyl groups can be removed by a protein encoded by our MGMT gene. However, the protein can only do it once, so the removal of each methyl group requires another molecule of protein.

This illustrates a problem with direct reversal mechanisms of DNA repair: they are quite wasteful. Each of the myriad types of chemical alterations to bases requires its own mechanism to correct. What the cell needs are more general mechanisms capable of correcting all sorts of chemical damage with a limited toolbox. This requirement is met by the mechanisms of excision repair.

Base Excision Repair (BER)

The steps and some key players:

1. removal of the damaged base (estimated to occur some 20,000 times a day in each cell in our body!) by a DNA glycosylase. We have at least 8 genes encoding different DNA glycosylases each enzyme responsible for identifying and removing a specific kind of base damage.
2. removal of its deoxyribose phosphate in the backbone, producing a gap. We have two genes encoding enzymes with this function.
3. replacement with the correct nucleotide. This relies on DNA polymerase beta, one of at least 11 DNA polymerases encoded by our genes.
4. ligation of the break in the strand. Two enzymes are known that can do this; both require ATP to provide the needed energy.

Nucleotide Excision Repair (NER)

NER differs from BER in several ways.

• It uses different enzymes.
• Even though there may be only a single "bad" base to correct, its nucleotide is removed along with many other adjacent nucleotides; that is, NER removes a large "patch" around the damage.

The steps and some key players:

1. The damage is recognized by one or more protein factors that assemble at the location.
2. The DNA is unwound producing a "bubble". The enzyme system that does this is Transcription Factor IIH, TFIIH, (which also functions in normal transcription).
3. Cuts are made on both the 3' side and the 5' side of the damaged area so the tract containing the damage can be removed.
4. A fresh burst of DNA synthesis — using the intact (opposite) strand as a template — fills in the correct nucleotides. The DNA polymerases responsible are designated polymerase delta and epsilon.
5. A DNA ligase covalent binds the fresh piece into the backbone.

Xeroderma Pigmentosum (XP)

XP is a rare inherited disease of humans which, among other things, predisposes the patient to

• pigmented lesions on areas of the skin exposed to the sun and
• an elevated incidence of skin cancer.

It turns out that XP can be caused by mutations in any one of several genes — all of which have roles to play in NER. Some of them:

• XPA, which encodes a protein that binds the damaged site and helps assemble the other proteins needed for NER.
• XPB and XPD, which are part of TFIIH. Some mutations in XPB and XPD also produce signs of premature aging. [Link]
• XPF, which cuts the backbone on the 5' side of the damage
• XPG, which cuts the backbone on the 3' side.

Transcription-Coupled NER

Nucleotide-excision repair proceeds most rapidly

• in cells whose genes are being actively transcribed
• on the DNA strand that is serving as the template for transcription.

This enhancement of NER involves XPB, XPD, and several other gene products. The genes for two of them are designated CSA and CSB (mutations in them cause an inherited disorder called Cockayne's syndrome).

The CSB product associates in the nucleus with RNA polymerase II, the enzyme responsible for synthesizing messenger RNA (mRNA), providing a molecular link between transcription and repair.

One plausible scenario: If RNA polymerase II, tracking along the template (antisense) strand), encounters a damaged base, it can recruit other proteins, e.g., the CSA and CSB proteins, to make a quick fix before it moves on to complete transcription of the gene.

Mismatch Repair (MMR)

Mismatch repair deals with correcting mismatches of the normal bases; that is, failures to maintain normal Watson-Crick base pairing (A•T, C•G)

It can enlist the aid of enzymes involved in both base-excision repair (BER) and nucleotide-excision repair (NER) as well as using enzymes specialized for this function.

• Recognition of a mismatch requires several different proteins including one encoded by MSH2.
• Cutting the mismatch out also requires several proteins, including one encoded by MLH1.

Mutations in either of these genes predisposes the person to an inherited form of colon cancer. So these genes qualify as tumor suppressor genes.

Synthesis of the repair patch is done by the same enzymes used in NER: DNA polymerase delta and epsilon.

Cells also use the MMR system to enhance the fidelity of recombination; i.e., assure that only homologous regions of two DNA molecules pair up to crossover and recombine segments (e.g., in meiosis).

Repairing Strand Breaks

Ionizing radiation and certain chemicals can produce both single-strand breaks (SSBs) and double-strand breaks (DSBs) in the DNA backbone.

Single-Strand Breaks (SSBs)

Breaks in a single strand of the DNA molecule are repaired using the same enzyme systems that are used in Base-Excision Repair (BER).

Double-Strand Breaks (DSBs)

There are two mechanisms by which the cell attempts to repair a complete break in a DNA molecule:

• Direct joining of the broken ends. This requires proteins that recognize and bind to the exposed ends and bring them together for ligating. They would prefer to see some complementary nucleotides but can proceed without them so this type of joining is also called Nonhomologous End-Joining (NHEJ).

·         Errors in direct joining may be a cause of the various translocations that are associated with cancers.

• Examples:
• Burkitt's lymphoma
• the Philadelphia chromosome in chronic myelogenous leukemia (CML)
• B-cell leukemia
• Homologous Recombination. Here the broken ends are repaired using the information on the intact
• sister chromatid (available in G₂ after chromosome duplication), or on the
• homologous chromosome (in G₁; that is, before each chromosome has been duplicated). This requires searching around in the nucleus for the homolog — a task sufficiently uncertain that G₁ cells usually prefer to mend their DSBs by NHEJ. or on the
• same chromosome if there are duplicate copies of the gene on the chromosome oriented in opposite directions (head-to-head or back-to-back). [Example]

Two of the proteins used in homologous recombination are encoded by the genes BRCA1 and BRCA2. Inherited mutations in these genes predispose women to breast and ovarian cancers.

Meiosis also involves DSBs

Recombination between homologous chromosomes in meiosis I also involves the formation of DSBs and their repair. So it is not surprising that this process uses the same enzymes.

Meiosis I with the alignment of homologous sequences provides a mechanism for repairing damaged DNA; that is, mutations. in fact, many biologists feel that the main function of sex is to provide this mechanism for maintaining the integrity of the genome.

However, most of the genes on the human Y chromosome have no counterpart on the X chromosome, and thus cannot benefit from this repair mechanism. They seem to solve this problem by having multiple copies of the same gene — oriented in opposite directions. Looping the intervening DNA brings the duplicates together and allowing repair by homologous recombination.

Gene Conversion

If the sequence used as a template for repairing a gene by homologous recombination differs slightly from the gene needing repair; that is, is an allele, the repaired gene will acquire the donor sequence. This nonreciprocal transfer of genetic information is called gene conversion.

The donor of the new gene sequence may by:

• the homologous chromosome (during meiosis)
• the sister chromatid (also during meiosis)
• a duplicate of the gene on the same chromosome (during mitosis)

Gene conversion during meiosis alters the normal mendelian ratios. Normally, meiosis in a heterozygous (A,a) parent will produce gametes or spores in a 1:1 ratio; e.g., 50% A; 50% a. However, if gene conversion has occurred, other ratios will appear. If, for example, an A allele donates its sequence as it repairs a damaged a allele, the repaired gene will become A, and the ratio will be 75% A; 25% a.

Cancer Chemotherapy

• The hallmark of all cancers is continuous cell division.
• Each division requires both
• the replication of the cell's DNA (in S phase) and
• transcription and translation of many genes needed for continued growth.
• So, any chemical that damages DNA has the potential to inhibit the spread of a cancer.
• Many (but not all) drugs used for cancer therapy do their work by damaging DNA.

• the cancer patient has many other cell types that are also proliferating rapidly, e.g., cells of the
• intestinal endothelium
• bone marrow
• hair follicles

and anticancer drugs also damage these — producing many of the unpleasant side effects of "chemo".

Agents that damage DNA are themselves carcinogenic, and chemotherapy poses a significant risk of creating a new cancer, often leukemia.

********************* 

Reference Books:

1.      De Robertis, E.D.P. and De Robertis, E.M.F., 2001, Cell and Molecular Biology, Lippincott Williams and Wilkins, USA. 

2.      Gupta, P.K., 1999, Cell and Molecular Biology, Rastogi Publications, Meerut.

3.      Pavlella. P., 1998, Introduction to Molecular Biology, McGraw-Hill Companies Inc., New York.

4.      Roy, S.C and De, K.K, 2001, Cell Biology,  New Central Book Agency, Calcutta

5.      Cell Biology, Genetics, Molecular Biology, Evolution and Ecology by P.S. Verma and V.K. Agarwal – S. Chand & Co.

*****************