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UNIT 1

CENTRAL DOGMA OF MOLECULAR GENETICS The central dogma is the original proposal that DNA makes RNA makes protein, which happen via the processes of transcription and translation respectively. This is broadly correct, although a number of examples are known which contradict parts of it. Retroviruses reverse transcribe RNA into DNA, a number of viruses are able to replicate RNA directly into an RNA copy, and a number of organisms can edit a messenger RNA sequence so that the protein coding sequence is not directly specified by DNA sequence. Following is the flow diagramme of central dogma -

However, a few exceptions to this basic scheme have been identified. A number of classes of virus contain a genome consisting of an RNA molecule. In the retroviruses, which include HIV, the causative agent of AIDS, the single-stranded RNA molecule is converted to a double-stranded DNA copy , which is then inserted into the genome of the host cell. This process has been termed reverse transcription, and the enzyme responsible, which is encoded in the viral genome, is called reverse transcriptase. There are also a number of viruses known whose RNA genome is copied directly into RNA, without the use of DNA as an intermediary (RNA replication). Examples include the coronaviruses and hepatitis C virus. As far as is known, there are no examples of a protein being able to specify a specific RNA or DNA sequence, so the translation step of the central dogma does appear to be unidirectional. Reference- Turner. Phil.Mclennan. Alexander, Bates. Andy,White. Mike, Instant Notes on Molecular Biology (2003), 3rd edition, Taylor & Francis Group

STRUCTURE OF DNA

The two common WatsonCrick base pairs of DNA. Adenine (A) is joined to thymine (T) by two hydrogen bonds, while guanine (G) is joined to cytosine (C) by three hydrogen bonds. Watson and Crick model of DNA

http://www.scribd.com/doc/49404744/DNA-and-RNA-structure-best B-DNA (WatsonCrick DNA) B-DNA is a right-handed helix; it turns in a clockwise manner when viewed down its axis. The bases are stacked almost exactly perpendicular to the main axis with 10.5 bases per turn. The major groove is wide and of moderate depth, while the minor groove is of moderate depth but is much narrower. B-DNA occurs under conditions of high humidity (95%) and relatively low salt. Since the inside of a cell is mostly water with relatively low salt concentration, it follows that the predominant form in vivo is B-DNA. A-DNA If the water content is decreased and the salt concentration increased during crystal formation, the A form of DNA (A-DNA) will occur. In this right-handed helix the bases are tilted with respect to the axis and there are more (11) bases per turn than in BDNA. The major groove of A-DNA is deep and narrow, while the minor groove is shallow and broad. It is unlikely that A-DNA is present in any lengthy sections in cells. However, RNA adopts an A-form helix when it forms double-stranded regions. The 2-hydroxyl group on the ribose sugar hinders formation of B-form RNA . Z-DNA In 1979, Alexander Rich and his colleagues at the Massachusetts Institute of Technology (MIT) made a novel discovery. They found that oligonucleotides composed of repeating GC sequences on one strand, with the complementary CG sequences on the other, formed a left-handed helix. A left-handed helix turns counter clockwise away from the viewer when viewed down its axis. Because the backbone formed a zig-zag structure, they called the structure Z-DNA. Z-DNA has 12 base pairs per turn. The minor groove is very deep and narrow. In contrast, the major

groove is shallow to the point of being virtually nonexistent. ZDNA was first formed under conditions of high salt or in the presence of alcohol. Later, it was shown that this form of double helix can be stabilized in physiologically normal conditions, if methyl groups are added to the cytosines. Reference- Lizabeth A. Allison (2007) Fundamental Molecular Biology BLACKWELL PUBLISHING 350 Main Street, Malden, MA 02148-5020, USA 9600 Garsington Road, Oxford OX4 2DQ, UK 550 Swanston Street, Carlton, Victoria 3053, Australia

WHY DNA REPLICATION IS SEMICONSERVATIVE? Daughter stand obtained after replication of DNA consists of one parental strand i.e. old strand and one newly synthesized strand so it is called as semiconservative . It was first proved by Messelsen and Stahl by using isotopes of nitrogen and CsCl2 gradient centrifugation method .

The mode of replication was determined in 1958 by Matthew Meselson and Franklin W. Stahl. They designed an experiment to distinguish between semiconservative, conservative, and dispersive replication. First, they needed a way to tell original DNA from newly synthesized DNA. To this end, they grew Escherichia coli in medium containing 15N, a heavy isotope of nitrogen. 15N contains one more neutron than the naturally occurring 14N. Unlike radioisotopes, 15N is stable and is not radioactive. After growing several generations of bacteria in the 15N medium, the DNA of E. coli became denser because the nitrogenous bases had incorporated the heavy isotope. The density of the strands

was determined using a technique known as density-gradient centrifugation. A solution of cesium chloride (CsCl) a heavy metal salt containing the DNA samples is spun in an ultracentrifuge at high speed for several hours. Eventually, equilibrium between centrifugal force and diffusion occurs, such that a gradient forms with a high concentration of CsCl at the bottom of the tube and a low concentration at the top. DNA forms a band in the tube at the point where its density is the same as that of the CsCl. The bands are detected by observing the tubes with ultraviolet light at a wavelength of 260 nm, in which DNA absorbs strongly. After many generations, Meselson and Stahl transferred the bacteria with heavy (15N) DNA to a medium containing only 14N. What they found was that DNA replicated in the 14N medium was intermediate in density between light (14N) and heavy (15N). In the next generation, only DNA of intermediate and light density was present. If replication had been conservative, there would have been two bands at the first generation of replication helix. Additionally, throughout the experiment, the original DNA would have continued to show up as a 15N (heavy) band. If the method of replication had been dispersive, the result would have been various multiple-banded patterns, depending on the degree of dispersiveness. Reference- Lizabeth A. Allison (2007) Fundamental Molecular Biology BLACKWELL PUBLISHING 350 Main Street, Malden, MA 02148-5020, USA 9600 Garsington Road, Oxford OX4 2DQ, UK 550 Swanston Street, Carlton, Victoria 3053, Australia

Proof of DNA as genetic material Grifith (1928) performed transformation experiment by using virulent and non virulent strain of Diplococcus pneumonia in mice but he could not explained the reason why mice injected with mixture of dead virulent strain and live non virulent strain die. Later on group of researcher of Avery, Mccarty and Macleoid (1944) was able to explain it and by that way it was proved that DNA is genetic material .

Reference- Lizabeth A. Allison (2007) Fundamental Molecular Biology BLACKWELL PUBLISHING 350 Main Street, Malden, MA 02148-5020, USA 9600 Garsington Road, Oxford OX4 2DQ, UK 550 Swanston Street, Carlton, Victoria 3053, Australia Transcription Transcription is the synthesis of RNA under the direction of DNA. RNA synthesis, or transcription, is the process of transcribing DNA nucleotide sequence information into RNA sequence information. Both nucleic acid sequences use complementary language, and the information is simply transcribed, or copied, from one molecule to the other. DNA sequence is enzymatically copied by RNA polymerase to produce a complementary nucleotide RNA strand, called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell. One significant difference between RNA and DNA sequence is the presence of U, or uracil in RNA instead of the T, or thymine of DNA. In the case of proteinencoding DNA, transcription is the first step that usually leads to the expression of the genes, by the production of the mRNA intermediate, which is a faithful transcript of the gene's protein-building instruction. The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. A DNA transcription unit that is translated into protein contains sequences that direct and regulate protein synthesis in addition to coding the sequence that is translated into protein. The regulatory sequence that is before (upstream (-) , towards the 5' DNA end) the coding sequence is called 5' untranslated region (5'UTR), and sequence found following (downstream (+), towards the 3' DNA end) the coding sequence is called 3' 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, RNA is synthesized in the 5' 3' direction (from the point of view of the growing RNA transcript). Only one of the two DNA strands is transcribed. This strand is called the template strand, because it provides the template for ordering the sequence of nucleotides in an RNA transcript. The other strand is called the coding strand, because its sequence is the same as the newly created RNA transcript (except for uracil being substituted for thymine). The DNA template strand is read 3' 5' by RNA polymerase and the new RNA strand is synthesized in the 5' 3' direction. A polymerase binds to the 3' end of a gene (promoter) on the DNA template strand and travels toward the 5' end. Prokaryotic and Eukaryotic Transcription 1. Prokaryotic transcription occurs in the cytoplasm alongside translation.

2. Eukaryotic transcription is localized to the nucleus, where it is separated from the cytoplasm by the nuclear membrane. The transcript is then transported into the cytoplasm where translation occurs. 3. Another important difference is that eukaryotic DNA is wound around histones to form nucleosomes and packaged as chromatin. Chromatin has a strong influence on the accessibility of the DNA to transcription factors and the transcriptional machinery including RNA polymerase. 4. In prokaryotes, mRNA is not usually modified. Eukaryotic mRNA is modified through RNA splicing, 5' end capping (5' cap), and the addition of a polyA tail. Transcription is divided into 5 stages: pre-initiation, initiation, promoter clearance, elongation and termination. Pre-Initiation Unlike DNA replication, transcription does not require primers for initiation. However RNA polymerase does require the presence of a core promoter sequence in the DNA, which it is able to bind to in the presence of various specific transcription factors. Promoters are regions of DNA which promote transcription and are found around 10 to -35 bp upstream from the start site of transcription. Core promoters are sequences within the promoter which are essential for transcription initiation. The most common type of core promoter in eukaryotes is a TATA box, with a consensus sequence of TATA(A/T)A(A/T). 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 what is known as the preinitiation complex. One such transcription factor 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 this pre-intiation complex. Other proteins known as activators and repressors, along with any associated co-activators or corepressors, may further enhance or inhibit transcription. Initiation

Simple diagram of transcription initiation. RNAP = RNA polymerase In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA. The RNA polymerase is a core enzyme consisting of five subunits: 2 subunits, 1 subunit, 1 ' subunit, and 1 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 basepairs downstream of promoter sequences. Transcription initiation is far more complex in eukaryotes, the main difference being that eukaryotic polymerases do not directly recognize their core promoter sequences. In eukaryotes, 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, called transcription initiation complex. Transcription in archaea 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. Once the transcript reaches approximately 23 nucleotides it no longer slips and elongation can occur. This is an ATP dependent process. Promoter clearance coincides with phosphorylation of serine 5 on the carboxy terminal domain of RNA Pol in prokaryotes, which is phosphorylated by TFIIH. Elongation

Simple diagram of transcription 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 is usually used as the reference point, so transcription is said to go from 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 produced from a single copy of a gene. This step also involves a proofreading mechanism that can replace incorrectly incorporated bases. Prokaryotic elongation starts with the "abortive initiation cycle". During this cycle RNA Polymerase will synthesize mRNA fragments 2-12 nucleotides long. This continues to occur until the factor rearranges, which results in the transcription elongation complex (which gives a 35 bp moving footprint). The factor is released before 80 nucleotides of mRNA are synthesized. In Eukaryotic transcription the polymerase can experience pauses. These pauses may be intrinsic to the RNA polymerase or due to chromatin structure. Often the polymerase pauses to allow appropriate RNA editing factors to bind. Termination

Simple diagram of transcription 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 well understood. It involves cleavage of the new transcript, followed by template-independent addition of As at its new 3' end, in a process called polyadenylation Source- http://www.scribd.com/doc

TRANSLATIONFollowing are the steps involved in protein translation in eukaryotes Scanning- Since there is no ShineDalgarno sequence in eukaryotic mRNA, the mechanismof selecting the start codon must be different. Kozak proposed a scanning hypothesis in which the 40S subunit, already containing the initiator tRNA, attaches to the 5_-end of the mRNA and scans along the mRNA until it finds an appropriate AUG. This is not always the first one as it must be in the correct sequence context (5_-GCCRCCAUGG-3_), where R = purine. Initiation - The overall mechanism of initiation involves four major steps: (i) the assembly of the 43S pre-initiation complex via a multifactor complex (MFC); (ii) recruitment of the 43S pre-initiation complex by the mRNA through interactions at its 5_-end; (iii) scanning to find the initiation codon; and (iv) recruitment of the 60S subunit to form the 80S initiation complex. There are at least 12 reasonably well defined initiation factors involved in eukaryotic protein synthesis, and some have analogous functions to the three prokaryotic IFs. They can be grouped in various ways but it is logical to group them according to the steps at which they act as follows: those involved in assembly of the 40S pre-initiation complex, such as eIF1, eIF1A, eIF2, eIF3 and eIF5; those binding to the mRNA to recognize the 5_-cap and to melt secondary structure such as eIF4B and eIF4F. eIF4F is a heterotrimer complex of an RNA helicase called eIF4A, a cap binding protein called eIF4E, and a scaffold protein, eIF4G; those that recruit the 60S subunit by displacing other factors such as eIF5B which releases 5 other factors so the 60S subunit can bind. The following events take place, starting with the eIF2-GTP binary complex that is formed by eIF2B recycling of the eIF2-GDP that is released late during initiation. The initiator tRNA joins to make a complex of three components (ternary complex), the initiator tRNA, eIF2 and GTP. In yeast, and probably other eukaryotes, the ternary complex then forms part of a multifactor complex (MFC) containing eIF1, eIF2-GTP-tRNAi, eIF3 and eIF5. The binding of the MFC to a free 40S subunit is assisted by eIF1A and the resulting complex is called the 43S pre-initiation complex. Note this different order of assembly in eukaryotes where the initiator tRNA is bound to the small subunit before the mRNA binds . Before this large complex can bind to the mRNA, the latter must have interacted with eIF4B and eIF4F (which recognizes the 5_-cap via eIF4E), and using energy from ATP, have been unwound and have had secondary structure removed by eIF4A. eIF4H may help in this. The second major step occurs when the 43S pre-initiation complex has bound to the mRNA complex via the interactions between eIF4G and eIF3. In the third step, ATP is used as the mRNA is scanned to find the AUG start codon. This is usually the first one. In the fourth step, to allow the 60S subunit to bind, eIF5B must displace eIF1, eIF2, eIF3 and eIF5 and GTP is hydrolyzed. eIF1A and eIF5B are released when the latter has assisted 60S subunit binding to form the complete 80S initiation complex. The released eIF2.GDP complex is recycled by eIF2B and the rate of recycling (and hence the rate of initiation of protein synthesis) is regulated by phosphorylation of the -subunit of eIF2. Certain events, such as viral infection and the resultant production of interferon, cause an inhibition of protein synthesis by promoting phosphorylation

of eIF2. Another point of regulation involves eIF4E binding/inhibitory proteins that can block complete assembly of eIF4F on some mRNAs . Elongation -The protein synthesis elongation cycle in prokaryotes and eukaryotes is quite similar. Three factors are required with similar properties to their prokaryotic counterparts . eEF1, eEF1 and eEF2 have the roles described for EF Tu, EF-Ts and EF-G respectively . Termination- In eukaryotes a single release factor, eRF, recognizes all three stop codons and performs the roles carried out by RF1 (or RF2) plus RF3 in prokaryotes. eRF requires GTP for activity, but it is not yet clear whether there is a eukaryotic equivalent of RRF required for dissociation of the subunits from the mRNA. There are surveillance mechanisms that operate in eukaryotes to release stalled ribosomes or degrade defective mRNAs. Nonstop mediated decay releases stalled ribosomes that have translated an mRNA lacking a stop codon. The translation product will be defective in that it will have polylysine at its carboxy terminus due to translation of the poly(A) tail and the ribosome is stuck at the 3_-end. A protein factor (Ski7) helps to dissociate the ribosome and recruit a 3_ to 5_ exonuclease to degrade the defective mRNA. Such polylysine tagged proteins are also rapidly degraded. In nonsense mediated mRNA decay (NMD) mRNAs containing premature stop codons are recognized due to the presence of protein complexes deposited at exonexon junctions following splicing in the nucleus . In normal mRNAs, these complexes are displaced by the first ribosome to translate the mRNA and the stop codon is reached later. In defective mRNAs, the premature stop codon is reached before all the complexes are displaced and this causes decapping of the mRNA and consequential 5_ to 3_ degradation. Reference- Turner. Phil.Mclennan. Alexander, Bates. Andy,White. Mike, Instant Notes on Molecular Biology (2003), 3rd edition, Taylor & Francis Group

GENETIC CODE The genetic code is the correspondence between the sequence of the four bases in nucleic acids and the sequence of the 20 amino acids in proteins. It has been shown that the code is a triplet code, where three nucleotides encode one amino acid, and this agrees with mathematical argument as being the minimum necessary [(42 = 16) _20 _(43= 64)]. However, since there are only 20 amino acids to be specified and potentially 64 different triplets, most amino acids are specified by more than one triplet and the genetic code is said to be degenerate, or to have redundancy. From a fixed start point, each group of three bases in the coding region of the mRNA represents a codon which is recognized by a complementary triplet, or anticodon, on the end of a particular tRNA molecule. The triplets are read in nonoverlapping groups and there is no punctuation between the codons to separate or delineate them. They are simply decoded as adjacent triplets once the process of decoding has begun at the correct start point . As more gene and protein sequence information has been obtained, it has become clear that the genetic code is very

nearly, but not quite, universal. This supports the hypothesis that all life has evolved from a single common origin. Features of Genetic code The genetic code is degenerate (or it shows redundancy). This is because 18 out of 20 amino acids have more than one codon to specify them, called synonymous codons. Only methionine and tryptophan have single codons. The synonymous codons are not positioned randomly, but are grouped in the table. Generally they differ only in their third position. In all cases, if the third position is a pyrimidine, then the codons specify the same amino acid (are synonymous). In most cases, if the third position is a purine the codons are also synonymous. If the second position is a pyrimidine then generally the amino acid specified is hydrophilic. If the second position is a purine then generally the amino acid specified is polar.

Reference- Turner. Phil.Mclennan. Alexander, Bates. Andy,White. Mike, Instant Notes on Molecular Biology (2003), 3rd edition, Taylor & Francis Group CHARGAFFs RULE

The three regular relationships among the molar concentrations of the different bases in DNA: (1) The number of A residues in all DNA samples is equal to the number of T residues; i.e. [A] = [T], where the molar concentration of the base is denoted by the symbol for the base enclosed in square brackets. (2) The number of G residues equals that of C; i.e. [G] = [C]. (3) The amount of purine bases equals that of the pyrimidine bases: [A] + [G] = [T] + [C]. Reference- Lizabeth A. Allison (2007) Fundamental Molecular Biology BLACKWELL PUBLISHING 350 Main Street, Malden, MA 02148-5020, USA 9600 Garsington Road, Oxford OX4 2DQ, UK 550 Swanston Street, Carlton, Victoria 3053, Australia TRANSFER RNA

tRNAs are RNA molecules that provide the means of translating the genetic code. One end of the tRNA contains a three nucleotide sequence called the anticodon loop that is complementary to the codon of the mRNA. The other end of the tRNA is covalently attached to a specific amino acid. Transfer RNAs (tRNAs), the smallest of the three major species of RNA molecules (4S), have between 74 and 95 nucleotide residues. tRNAs species make up about 15 % of the total RNA in the cell. The tRNA molecules contain unusual bases e.g. dihydrouracil, and have extensive intrachain base-pairing (Figure 1). Each tRNA serves as an 'adaptor" molecule that carries its specific amino acid-covalently attached to its 3 end-to the site of protein synthesis.

A. Characteristic t RNA structure B. Folded tRNA structure Source- http://www.scribd.com/doc

EUKARYOTIC mRNA Messenger RNAs are RNA molecules that carry the "message" from the DNA to the ribosomes to be translated into protein. The "message" in mRNA is carried in groups of three nucleotides called codons.

Each codon specifies one amino acid in a protein according to the rules of the genetic code. Messenger RNA (mRNA) comprises only about 5 % of RNA in the cell. It is the most heterogeneous type of RNA in size (500 to 6000 nucleotides) and base sequence.

The mRNA carries genetic information from the nuclear DNA to the cytosol where it is used as the template for protein synthesis. Special structural characteristics of eukaryotic mRNA (but not prokaryotic) include a long sequence of adenine nucleotides (a 'poly-A tail) on the 3 -end of the RNA chain plus a 'cap' on the 5 -end consisting of a molecule of 7-methylguanosine attached 'backward' (5'5') through a triphosphate linkage

EUKARYOTIC RIBOSOME The ribosomes are oblate spheroid structures of 150 to 250Ao in diameter. Each ribosome is porous, hydrated and composed of two subunits. One ribosomal subunit is large in size and has a domelike shape, while the other ribosomal subunit is smaller in size and occurring above the larger subunit and forming a cap-like structure. The 80S ribosome also consists of two subunits, viz., 60S and 40S. The 60S ribosomal subunit is dome-shaped and larger in size. In the ribosomes which remain attached with the membranes of endoplasmic reticulum and nucleus, etc., the 60S subunit remains attached with the membranes. The 40S ribosomal subunit is smaller in size and occurs above the 60s subunit forming a cap-like structure. Both the subunits remain separated by a narrow cleft . The two ribosomal subunits remain united with each other due to high concentration of the Mg++(.001M) ions. When the concentration of Mg++ions reduces in the matrix, both ribosomal subunits get separated. Actually in bacterial cells the two subunits are found to occur freely in the cytoplasm and they unite only during the process of protein synthesis. At high concentration of Mg++ ions in the matrix, the two ribosomes (called monosomes) become associated with each other and known as the dimer. Further, during protein synthesis many ribosomes are aggregated due to common messenger RNA and form the polyribosomes or polysomes. Function of ribosome is synthesis of protein by using mRNA template with the help of tRNA and amino acid.

REFERENCETurner. Phil.Mclennan. Alexander, Bates. Andy,White. Mike, Instant Notes on Molecular Biology (2003), 3rd edition, Taylor & Francis Group Lizabeth A. Allison (2007) Fundamental Molecular Biology BLACKWELL PUBLISHING 350 Main Street, Malden, MA 02148-5020, USA 9600 Garsington Road, Oxford OX4 2DQ, UK 550 Swanston Street, Carlton, Victoria 3053, Australia Verma. P k, agarwal. V k,(2005). Cell biology, Genetics, Molecular biology, Evolution and Ecology, s. Chand & company ltd. Ram nagar, new delhi-110 055

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