Molecular Genetics Principles of Gene Expression: Transcription

Transcription

1 Paper No. : 16 Module : 12 Principles of gene expression: Transcription Development Team Principal Investigator: Prof. Neeta Sehgal Head, Department of Zoology, University of Delhi Paper Coordinator: Prof. Namita Agrawal Department of Zoology, University of Delhi Content Writer: Dr. Sudhida Gautam, Hansraj College, University of Delhi Dr. Kiran Bala, Deshbandhu College, University of Delhi Content Reviewer: Dr. Surajit Sarkar, Department of Genetics, South Campus, Delhi University 1

2 Description of Module Subject Name Paper Name ; Zool 016 Module Name/Title Module Id Keywords 12; Transcription Central dogma, Transcription, RNA polymerase, template, Pribnow nox, promoter, splicing, holoenzyme, monocistronic Contents 1. Learning Outcomes 2. Introduction 3. Transcription 3.1 Components 3.2 Types of RNA 4. Experimental Evidences 4.1 DNA acts as template for transcription 4.2 One DNA strand acts as a template 5. Transcription Unit 6. RNA Polymerase 6.1 Bacterial RNA polymerase 6.2 Eukaryotic RNA polymerase 7. Bacterial Transcription 7.1 Initiation 7.2 Elongation 7.3 Termination 8. Transcription in Eukaryotes 8.1 Initiation 8.2 Elongation 8.3 Termination (i) Allosteric model (ii) Torpedo model 9. Post Transcriptional Modifications 9.1 RNA splicing 9.2 Pre-mRNA Processing/ 3 and 5 modifications 10. Summary 2

3 1. Learning Outcomes The following module explains the central dogma of molecular biology. The basic differences of DNA and RNA. Purpose of transcription process in biological system and the three events (initiation, elongation and termination). How the genetic information which is coded in the DNA is converted to RNA before translation can begin. Differences between prokaryotic and eukaryotic transcription during the three events (initiation, elongation and termination) 2. Introduction The word transcript means written or printed version of something. Transcription is a vital process of the biological forms having a complex regulatory system. In 1953 Watson and Crick gave the double helix model of DNA, and three years later Crick gave the Central Dogma of molecular biology (Figure 1) which stated that genetic flow of information within the different organism s is a two step process. The genetic information stored in DNA is activated to give mrna (Transcription) and from mrna finally to proteins (Translation). Figure 1: The central dogma of molecular biology Table 1 tells us about the differences between molecule of DNA and RNA. The carbon at second position varies for DNA and RNA, absence of oxygen in the sugar at C2 gives it the name as deoxyribose sugar (Figure2). Table 1: Difference between RNA and DNA Characteristic DNA (Deoxyribose Nuclei acid) RNA (Ribose Nuclei acid) Sugar Deoxyribose sugar Ribose sugar Nucleotides A,T,G,C A,U,G,C Strands Double stranded Single stranded Presence of 2 -OH group (Figure 2) No Yes Stability More stable Less stable Types A,B and Z rrna, mrna, trna, SnRNA, mirna, sirna 3

4 3. Transcription (A) Ribose Sugar (B) Deoxyribose sugar Figure 2: Structure of Pentoses sugar: (A) Ribose; (B) Deoxyribose Source: The genetic code of the DNA (genotype) is passed to RNA through the process of transcription. It requires a series of events involving varies RNA nucleotides, DNA template and a series of protein components for its initiation and regulation Components The transcription requires three major components: 1. RNA polymerase and associated protein factors 2. DNA template 3. Raw materials (Nucleotides) RNA polymerase catalyzing the process of transcription was discovered independently in 1960 by Samuel Weiss and Jerard Hurwitz. The information on the DNA is used by RNA polymerase to make mrna using one of the strands of DNA as a template also known as the anti-sense or non-coding strand. The double stranded DNA is unwound, one strand to which the RNA polymerase attaches acts as the template strand (Figure 3). The other strand is referred to as the non-template strand, coding strand or sense strand. Transcription begins on specific DNA sequences called promoters. It occurs in three phases initiation, elongation and termination. 4

5 Figure 3: A transcribing unit Source: ( Types of RNA RNA (Ribose nucleic acid) is a polymer of ribonucleotides linked together by 3-5 phosphodiester bond. To begin the chapter we ll first have a brief discussion about the different types of RNA (Table 2). Table 2: Types of RNA: Depending upon the type of function the RNA molecules are classified as- Type of RNA Function Location Ribosomal RNA (rrna) Structural component of the ribosomes Cytoplasm Messenger RNA (mrna) Transfer RNA (trna) Carries the information in a gene for the protein synthesis Transport amino acids to the ribosomes during protein synthesis Nucleus and cytoplasm Cytoplasm Small nuclear RNA (snrna) Modification of the RNA transcript Nucleus Micro RNA (mirna) RNA interference Nucleus Small interfering RNA( Si RNA) RNA interference Nucleus 5

6 4. Experimental Evidences/Historical Studies Helpful to Study Transcription 4.1. DNA acts as the template for transcription In 1970, Oscar miller, Barbara Hamkalo and Charles Thomas provided the evidence of DNA molecule being used as a template for transcription. Electron microscopic studies of internal cellular contents revealed presence of Christmas-tree like structures; thin central fibers (the trunk of the tree), to which were attached strings (the branches) with granules (Figure 4). When deoxyribonucleases were added to it breakdown of the central fibers was observed, indicating that the tree trunk were DNA molecules. Ribonucleases removed the granular strings, indicating that the branches were RNA. The Christmas tree like structures was concluded to be a gene undergoing transcription. As, the process of transcription proceeds, more and more RNA is formed which further extends the branches of the tree. (a) Christmas tree (b) RNA Polymerase and DNA chain Figure 4: Christmas tree like structures within the cell showing the site of transcription Source: (a) (b) DNA molecule unwinds to act as a template i.e. the double helix opens up and only one strand acts as template for the transcription. This is the strand to which the RNA polymerase binds and transcription process continues which is exactly opposite to the coding strand which contains the gene sequences. As transcription takes place on this template the new strand is perfect replica of the coding strand of DNA molecule. At a given time only one of the DNA strand acts as a template One strand of DNA acts as a template for transcription In 1963, Julius Marmur and his colleagues (Figure5) proved that only one strand acts as template. They made use of DNA of bacteriophage SP8, which infects the Bacterium Bacillus subtilis. The double stranded DNA of this phage has different densities for each strand, which permits the separation of the two strands by equilibrium density gradient configuration into "heavy" and "light" DNA strands. 6

7 B. subtilis was placed in a medium containing a radioactively labeled precursor of RNA by Marmur and his colleagues. Later the bacteria were infected with SP8, as a result the phage DNA was injected into the bacterial cells. Transcription within the infected bacterial cells produced radioactively labeled RNA complementary to the phage DNA. This newly synthesized RNA was isolated from the cells. Secondly, DNA from fresh culture of SP8 was isolated and the two strands were separated into heavy and light stand of DNA, respectively. The radioactively labeled RNA obtained from the infected bacterial cells was combined with the heavy strand and the light strand. Hybridization was observed with the heavy strand.however, when radioactively labeled was combined with the light strand no hybridization took place. Hence, evidence was provided by Marmur and his colleagues which proved that RNA is transcribed from only one of the DNA strands in SP8. Heavy strand acted as the template in this case. The newly synthesized RNA strand was complementary and anti-parallel to this strand, having the same polarity and base sequences as that of the non-template strand, with the exception that T in DNA is replaced by U in RNA. Figure 5: Marmur s Experiment to prove only one strand of DNA acts as template during transcription 5. Transcription Unit A transcription unit (Figure 6) is a stretch of DNA or a particular gene sequence which is flanked by a promoter and terminator region, upstream of the start site and downstream of the terminator region respectively. The three components namely are: 7

8 1. Promoter: The DNA sequences (consensus sequences) located in the 5 region which promotes/recruits or initiates the transcription process. These consensus sequences have been conserved throughout the evolutionary process and share homology in different genes of the same organism or in one or more related organism. It is present, upstream of the RNA coding region/transcriptional start site. The promoter facilitates the binding of transcription apparatus to the DNA template and ensures that the initiation of each RNA occurs at the same point. Two promoter regions have been identified in the bacterial system; (a) Pribnow box/tata box located -10 upstream from the site of initial transcription (TATAAT sequence; rich in adenine and thymine) and (b) TTGACA located 35 nucletiodes upstream. The specific sequence of the promoter is responsible for the binding strength of the RNA polymerase to the transcription unit. 2. RNA coding sequence: The base pair sequence of the template DNA which is copied in the RNA molecule. 3. Terminator: The sequences of nucleotides which signals the termination of transcription and are part of the coding region. 6. RNA Polymerase Figure 6: A transcription unit Source: The enzyme RNA polymerase catalyzes the process of transcription 6.1. Bacterial RNA polymerase A single large multimeric, RNA polymerase catalyses the process of bacterial transcription (Figure 8). The RNA polymerase consists of a core enzyme made of 5 polypeptides which are two α, one β, and one β, bound to another polypeptide called the sigma factor (σ). The sigma factor recognizes the upstream -35 and -10 regions of the promoter and makes sure that this binding of the RNA polymerase and DNA is stable. Core enzyme can carry on the process on its own back its lacks the ability to bind at specific promoter region. The binding efficiency of the RNA polymerase varies according to the actual sequence of the promoter region. The polymerase binds to the promoter region while the DNA is still in the double helical form, known as the closed promoter complex. Holoenzyme is the actual functional enzyme which helps in the opening up of the DNA helix, by 8

9 melting a short stretch of the DNA helix. This untwisted form of the promoter is known as open promoter complex. The sigma factor can leave the transcription bubble once the RNA chain reaches 8-9 base pairs. The released sigma can be used for initiation by another RNA polymerase. Further elongation of the RNA chain can proceed without requiring the sigma factor. The core enzyme can bind to the DNA molecule with the same affinity at any position. The binding sites for core enzyme DNA are known as loose binding sites. But the holoenzyme binds to promoters very tightly, with an association constant increased from that of core enzyme by (on average) 1000 times and with a halflife of several hours Eukaryotic RNA polymerase RNA polymerase is large and complex enzymes e.g. yeast holoenzyme consists of two large subunits and ten small subunits (Figure7). Three different types of RNA polymerase are present in eukaryotes responsible for transcribing a different class of RNA (Table: 3). Compared to prokaryotic RNA these RNA polymerase are large multimeric units; thus several genes encode for them. Table 3: Types of eukaryotic RNA Polymerase RNA polymerase Function Location RNA polymerase I Transcribes 28S,18S and 5.8 S rrna molecules Nucleolus RNA polymerase II Transcribes mrna, sn RNA Nucleoplasm of the nucleus RNA polymerase III Transcribes trna, 5S rrna, some snrna and few mirna s molecules Nucleoplasm RNA polymerase IV Some sirna in plants Nucleus in plants 9

10 Figure 7: Comparison of structural composition of prokaryotic and eukaryotic RNA polymerase Source: 7. Bacterial Transcription The basic transcription unit and apparatus have already been discussed we ll study in detail how the process is carried on. To begin with transcription can be easily divided into three phases namely: 7.1. Initiation Initiation consists of binding of RNA polymerase to the DNA helix for RNA synthesis to begin. The transcription apparatus recognizes and binds to the promoter region which is identified by the sigma complex. The DNA of closed promoter complex melts to result in open promoter complex. The template strand is identified and accordingly the nucleotides are added. Rate of transcription varies for 10

11 different genes, depending on the varying affinity of the promoter and RNA polymerase. Two DNA sequences in most promoters of E. coli which have played a critical role in initiation of transcription are found upstream at -35(helps in initial recognition) and -10 (for the melting reaction to convert closed promoter complex into an open promoter complex) (Figure6 and Figure8). The short stretch of nucleotides ahead of the promoter is referred to as the consensus sequence. The two most common consensus sequence of the most bacterial promoters are the -35 region (the -35 box) is 5 TTGACA- 3 and -10 region (the -10 box, formerly called the Pribnow box; after David Pribnow its discoverer) is 5 TATAAT-3. The sigma factor associates with the core enzyme to form holoenzyme enzyme. This holoenzyme binds to the consensus sequence and strongly to the promoter at the -10 region, simultaneously accompanied by a local untwisting of about bp around the region. Thus, RNA polymerase orients itself to begin the transcription at +1. The polymerase pairs the base of the nucleotide triphosphate with the complimentary base present on the template DNA. No primer is required for this paring; the next coming nucleotide is bound to the 3 end of the first nucleotide with the release of a pyrophosphate. Since no phosphodiester bond forms at the 5 end it continues to have the three phosphate groups. Figure 8: Binding of RNA apparatus to DNA Source: 11

12 7.2. Elongation Once the long RNA is synthesized the sigma factor leaves the transcription bubble. The RNA polymerase undergoes conformation and is no longer able to bind to the consensus sequences. The core enzyme moves along the template joining nucleotides to the RNA molecule, in the process it untwists the DNA double helix downstream and then reanneals it (Figure9). The rate of RNA synthesis is lower than that of DNA synthesis and average of nucleotides per second are added. Topoisomeraes help in the uncoiling and recoiling of the DNA template as transcription proceeds. RNA polymerase has proof reading property which helps to remove any noncomplimentary base and continue the transcription. If the enzyme encounters a wrong base it goes back cleaves it and resumes the synthesis in the forward direction Termination The sequences which code the termination of the transcription are referred to as the terminator sequences. Termination includes detaching of the enzyme from the DNA template and the release of the newly synthesized RNA molecule (Figure 9). Two types of terminators are present in the bacterial system with or without an ancillary protein called Rho factor; namely Rho dependent (also, type II terminators) and Rho independent terminators (also, type I terminators) (Table 4). A polycistronic RNA is produced when a number of genes are transcribed in a single RNA; i.e. a single termination occurs at the end. Polycistronic RNA are absent in eukaryotes as each gene has its own initiation and termination site. Table 4: Differences between type I (Rho independent) terminators and type II (Rho dependent) terminators Type I (Rho independent) terminators Termination takes place in absence of rho factor Terminator consists of an inverted repeat sequence When transcribed the inverted repeat sequence forms a hair-pin like loop. The termination sequence is followed by a string of approximately 6 adenine nucleotide; their transcription produce a string of uracil nucleotide after the hair-pin loop. Formation of the hair-pin slows down the polymerase and the adenine-uracil nucleotides which follow it are relatively unstable. This destabilization of the DNA RNA pairing; results in the release of the RNA molecule. Type II (Rho dependent) terminators Termination takes place in the presence of rho factor Terminator lacks the AT string found in Rho dependent terminators. Terminator lacks the hair-pin loop. Rho has RNA binding and ATPase domains. Rho binds to the unstructured RNA (stretch of RNA upstream of terminator sequence which lacks any secondary structure) and moves towards the 3 end. Rho reaches the transcription bubble and its helicase activity unwinds thee RNA-DNA hybrid and stops transcription. 12

13 Figure 9: Transcription steps Source: 8. Transcription in Eukaryotes Transcription in eukaryotes is similar to that of prokaryotes. However, it involves three RNA polymerase which help in recognition of specific promoter regions. These promoters/activators/ 13

14 enhancers have two regions namely; 1) core promoter and 2) promoter proximal region/regulatory promoter region (present upstream of a gene sequence). The core promoter is located upstream of the initiation site and consists of -35 to-52 base pairs. The TATA box (also known as Goldberg-Hogness box, after its discoverer) is present-25 to -30 bp upstream of the start site and consensus sequence is TATAAA. These promoters facilitate the formation of initiation complex and affect the rate of transcription. The regulatory promoter are located upstream of the core promoter eg; CAAT box (5' GGCCAATCT 3'), GC box (GGGCGG) ( box centered at about -75 to -120) (Figure10). Any mutation which takes place in this region markedly decreases the rate of transcription, indicating there role in the efficiency of the initiation complex Initiation Figure 10: Sequence elements of a general eukaryotic promoter/gene Source: Eukaryotic_Promoter_Structure_for_RNA_Polymerase_II_files/image004.jpg To begin the transcription proper assembly of the RNA polymerase and the general transcription factors (GTFs) are required. The GTFs are specific for each RNA polymerase and are numbered according to the RNA polymerase for which they work. These GTFs have replaced the sigma factor of prokaryotes. The GTFs are represented as TFIIA, TFIIB, TFIID, TFIIE and TFIIG. The final alphabetical letter designates the individual factor (Figure11). TFIID is the initial committed complex which recognizes and binds to the TATA box with the help of its TBP (TATA-binding protein). TATA-binding protein binds the major groove of DNA which results in its bending and unwinding of the DNA helix. The binding of TFIID facilitates the bending which helps in the binding of TFIIB to TFIID followed by sequential binding of other GTFs (TFIIA, TFIIF accompanied with polymerase and finally TFIIE and TFIIH) and RNA polymerase to produce the initiation complex (Table 5). TFIIE and TFIIH bind to the RNA polymerase to form the pre-initiation complex. TFIIH acts as a helicases (breaks the bond between the doube stranded DNA) to form an open complex. Conformational changes within the DNA and polymerase result in unwinding of 10-15bp of DNA. The template DNA is placed on the active site resulting in the formation of the open initiation complex. TFIIH also hydrolyses ATP to phosphorylate the carboxy terminal domain (CTD) in RNA polymerase II. This phosphorylation 14

15 breaks the contact between the RNA polymerase II and TFIIB. As, a result TFIIB, TFIIE and TFIIH dissociate from RNA polymerase and it s free to proceed the elongation process. Table 5: Function of the general transcription factors General Transcription Factor TFIID (composed of TATA-binding proteins (TBP) and TBP-associated factors (TAFs) TFIIB TFIIH TFIIA TFIIF TFIIE Function in transcription Recognizes the TATA box in the promoter region (core promoter binding factor) Interacts with TBP of TFIID and stabilizes TBP-TATA complex, recruits binding of TFIIF- RNA polymerase complex Helicases activity for opening of the promoter complex, initiates transcription (Enzymatic activities of DNA Helicase and ATP kinase) and repairs DNA damage ( by nucleotide excision repair) Stabilizes TBP-DNA binding Binds to RNA polymerase and prevents it from binding to nonspecific DNA binding sites Helps in maintenance of initiation complex and switching to elongation process 15

16 Figure 11: Initiation in eukaryotes Source: 16

17 8.2. Elongation Once the initiation complex synthesis the promoter region many of the transcription factors are disassembled and can be used by other RNA polymerases. The RNA transcript has a length of bp which keeps on elongating as new nucleotides are being added in the 3 end. During the elongation process 8 RNA nucleotides remain base paired with the DNA template. The DNA-RNA duplex is bent at 90 between the jaw-like extensions of the enzyme. As the complex moves forward the unwound DNA is rewound and separate RNA transcript exits from it. Roger Kornberg and his colleagues were awarded Nobel Prize in 2006 for studying the process of transcription. He discovered Mediators responsible for mediating the interacting between the RNA polymerase II and regulatory transcription factors which bind to enhancers or silencers and serve as an interference between RNA polymerase II and many diverse regulatory signals Termination In eukaryotes the RNA transcription continues down the DNA template until it encounters a poly A sequence. The mrna transcription can even continue past this poly A site, in some cases even 100 or 1000 bp. The poly A consensus sequence i.e. AAUAAA is a string of adenine nucleotides which continues near the 3 end of the mrna. The addition of a tail of polyadenylic acid (poly A) to the 3' end of mrna is referred to as polyadenylation. Polyadenylation involves recognizing the processing site signal, (AAUAAA), and cleaving of the mrna to create a 3' OH terminal end to which poly A polymerase adds adenylate residues. Transcription via RNA polymerase II typically terminates about 500 to 2000 nucleotides downstream from the poly A signal. Two models have being proposed for termination process namely, (i) Allosteric model: After transcribing the poly A sequence, RNA polymerase and DNA template destabilize, which ultimately results in their dissociation. For poly A addition to the RNA, a number of proteins including cleavage stimulation factor (CPSF) protein, and two cleavage factor proteins (CFI and CFII), bind to and cleave the RNA. Then, the enzyme poly A polymerase (PAP) uses ATP as a substrate and catalyzes the addition of A nucleotides to the 3 end of the RNA to produce the poly (A) tail. During this process PAP is bound to CPSF. As, the poly (A) tail is synthesized, molecules of poly (A) binding protein II (PABII) bind to it. (ii) Torpedo model: It requires the Rat 1 exonuclease. Cleavage of the mrna results in a 5 end trailing out of the RNA polymerase (Figure12). To this free 5 end the Rat 1 attaches and cleaves the growing RNA by moving towards the 3 end. Rat1 is a 5-3 exonuclease i.e. it cuts the RNA from 5 end towards 3 end. Like a torpedo it devours the growing RNA and on reaching the RNA polymerase it disrupts the transcriptional machinery and terminates transcription. 17

18 Figure12: Termination of transcription in eukaryotes. (1. Synthesis of polya tail); 2. RNA is released which destabilizes the RNA polymerase and DNA complex; 3. Allosteric model: Due to destabilization DNA and RNA polymerase seperate; 4. from the growing RNA Rat1 exonuclease binds; 5. Binding leads to a torpedo like action which ferociously cleaves the RNA leading to separation of DNA and RNA polymerase) Source: E302B1B1E1FA2.png 9. Post Transcriptional Modifications Unlike prokaryotes (which have polycistronic mrna and require no post transcriptional modifications) the eukaryotic mrna are modified at both the ends. Also, all the genes are not collinear with the proteins that they code (When a continuous sequence of nucleotides in DNA encodes a continuous sequence of amino acids in a protein, the two are said to be collinear). In 1970 s it was discovered that the regions of DNA were much longer than RNA. When DNA and RNA were hybridized the hybrid of DNA-RNA showed looped structures whereas DNA-DNA molecule could match through the entire length. It was concluded that certain regions of DNA are absent from the RNA. This provided evidence that the eukaryotic genes consisted of coding and non-coding regions. The coding sequences i.e. exons are disrupted by non-coding introns. The term intron refers to the intervening sequences which do not code the amino acid sequences. Exons are the expressed sequences which are ligated to obtain a continuous coding mrna. The introns are removed and the exons are joined together before the mrna leaves the nucleus. This process of joining the exons is known as RNA splicing (Figure 13, 14). The mrna bears three sites for splicing to take place which 18

19 are; 5 consensus/splice site which begins with 5 GU and a branch point followed by 3 splice site which has AG3 end. Above which is located a branch point approximately 18 to 40 bp 9.1. RNA splicing RNA splicing involves the removal of introns and joining of the exons. An endonucleolytic cut is made at each end of an intron, the intron is removed, and the exon ends are rejoined. RNA ligase seals the exon ends to complete each splicing event. However, the precise excision of introns is much more complex and interesting in higher eukaryotes. These catalytic RNAs were referred to as ribozymes. Thomas Cech and his colleagues discovered in 1963 during a study of the ciliate protozoan Tetrahymena. Figure 13: Self splicing introns Source: Concepts of genetics; Klug and Cummings tenth edition. Pg:335 19

20 1. Group I: Self splicing introns which are present in some rrna genes. The self excision involves an interaction between a guanosine cofactor and the primary transcript (Figure 13). The 3 -OH group of guanosine is transferred to the nucleotide adjacent to the 5 end of the intron. Then this newly acquired 3 -OH group (of guanosine) on the left-hand of exon and the phosphate on the 3 end of the intron form a bond. The intron is spliced out and the two exon regions are ligated, leading to the mature RNA. 2. Group II: Self-splicing introns with a different mechanism than that of the group I, are present in the protein coding genes of mitochondria and chloroplasts. An autocatalytic reaction leads to excision of intron, which lacks guanosine as a cofactor. 3. Nuclear pre-mrna introns: Splicing takes place within a large complex known as spliceosome which consists of a pre mrna bound to snrna (small nuclear RNA) ranging from 107 to 210 nucleotides which associate with proteins to form snrnps (small nuclear ribonucleoprotein particles). Small nuclear RNAs (snrnas or snurps) are an essential component of the splicesomal complex and are located in the protein coding genes of eukaryotic cell. Being rich in uridine they are known as U1, U2.U6. There sequential binding results in the formation lariat which contains the removed introns (Figure 14).The splicing reactions proceed as described below: U1 binds to the 5 splice end. U2 binds to the branch point. Complex of U4, U5, U6 joins the splicesome and combines the U1 and U2. This causes the introns to loop and brings the exons closer. U1 and U4 snrnps dissociate resulting in activation of the splicesome complex. Active complex removes the introns (in the form of a lariat) and ligates the two exons. The branch point bond breaks and the linear intron is easily digested by the nuclear enzymes. The snrnps are released after ligating the exon and this process is followed for each intron molecule. 4. Transfer RNA introns: found in the trna genes and makes use of specialized enzymes to cut and reseal the RNA. In prokaryotic cell both transcription and translation can take place at same time, as both the processes are coupled with each other. Thus, mrna produced has no opportunity to be modified. However, in eukaryotes the site of transcription and translation are nucleus and cytoplasm, respectively. Changes are incorporated into the nascent mrna at both the 3 and 5 end of the molecule to protect the coding of the molecule. 20

21 Figure 14: Lariat formations in Spliceosome Source: Concepts of genetics; Klug and Cummings tenth edition. Pg:336 21

22 9.2. Pre-mRNA Processing/ 3 and 5 modifications As mrna has around bp, a capping enzyme adds a methylated guanine nucleotide to the 5 end by an unusual 5 to 5 linkage as opposed to the usual 5 to 3 linkage. The methyl group is added to the position 7 of the base making the base 7-methylguanine. This is referred to as capping and the presence of this cap helps in removal of introns in addition to providing stability to the mrna. The 5 cap is easily recognized by the ribosomes, which binds to it and initiates the translation process. Rarely, addition methyl residues may be attached to the bases of the second and third nucleotide. A sequence of about adenine nucleotide base pairs are added to the 3 end of the mrna, forming a poly (A) tail. These are added after the mrna is released from the polymerase and is known as polyadenalytion. Polyadenalytion provides stability to the RNA molecule (protects from exonucleases) and a longer time period to be available for the translation process. Poly A site having nucleotides upstream of the cleavage has a consensus sequence of AAUAAA. 10. Summary The genetic information is passed on to generations through the central dogma of biology i.e. from DNA to RNA (via transcription) and from RNA to proteins (via translation). In Prokaryotes both the process occurs in nucleus whereas in eukaryotes site of transcription is nucleus and that of translation is cytoplasm. Transcription is the synthesis of RNA from a DNA. The DNA unwinds and RNA polymerase synthesizes RNA along with certain general transcription factors. The entire process is divided into three major steps; namely initiation, elongation and termination. Prokaryotes: A single RNA polymerase catalyzes the polymerization of ribonucleoside 5 - triphosphates (NTPs) and the growing chain is always in the 5 to 3 direction. The specific promoters are recognized by the σ subunit. and initiates the binding of RNA polymerase. Core polymerase consists of two α, one β, and one β subunits, is fully capable of catalyzing the polymerization of NTPs into RNA. The enzymes move along the DNA to continue elongation of the growing RNA chain. The moving polymerase maintains an unwound region of about 17 base pairs and the entire transcription bubble is referred to as open promoter complex. The addition of nucleotides continues until the polymerase encounters a termination signal. Termination is of two types (a) Rho dependant: Protein factor Rho binds to the end of the RNA chain along the strand towards the open complexand shears the RNA transcript and all components dissociate; (b) Rho independant: Transcription of the GC-rich inverted repeat results in the formation of a segment of RNA that can form a stable stem-loop structure by complementary base pairing. The formation of such a self-complementary structure in the RNA disrupts its association with the DNA template and terminates transcription. 22

23 In Eukaryotes: Transcription involves three RNA polymerase which help in recognition of specific promoter regions located upstream of the initiation site and consists of -35 to-52 base pairs followed by the TATA box. The General transcription factors (GTFs) have replaced the sigma factor of prokaryotes. TFIID is the initial committed complex which recognizes and binds to the TATA box with the help of its TBP (TATA-binding protein). The binding of TFIID facilitates the sequential binding of other GTFs (TFIID followed by TFIIA, TFIIB, TFIIF accompanied with polymerase and finally TFIIE and TFIIH) and RNA polymerase to produce the initiation complex. The RNA transcript has a length of25-30bp which keeps on elongating as new nucleotides are being added in the 3 end. Polyadenylation involves recognizing the processing site signal, (AAUAAA), and cleaving of the mrna to create a 3' OH terminal end to which poly A polymerase adds adenylate residues Transcription via RNA polymerase II typically terminates about 500 to 2000 nucleotides downstream from the poly A signal. Allosteric model states that after transcribing the poly A sequence, RNA polymerase and DNA template destabilize, which ultimately results in their dissociation. Torpedo model requires the Rat 1 exonuclease which cleaves the growing RNA and on reaching the RNA polymerase. It disrupts the transcriptional machinery and terminates transcription. The eukaryotic genes consisted of coding and non-coding regions. The coding sequences i.e. exons are disrupted by non-coding introns. This process of joining the exons is known as RNA splicing. As mrna has around 20-30bp, a capping enzyme adds a methylated guanine nucleotide to the 5 end by an unusual 5 to 5 linkage as opposed to the usual 5 to 3 linkage. The methyl group is added to the position 7 of the base making the base 7-methylguanine. This is referred to as capping and the presence of this cap helps in removal of introns in addition to providing stability to the mrna. The 5 cap is easily recognized by the ribosome, which binds to it and initiates the translation process. 23