SYNTHESIS OF RNA: TRANSCRIPTION To Study 's Transcription in prokaryotes: Prokaryotic RNA polymerase, Fole of sigma factor, promoter, Initiation, elongation and termination + Transcription in Eukaryotes: Eukaryotic RNA polymerases, transcription factors, promoters, enhancers, mechanism of transcription initiation, promoter clearance and elongation RNA splicing ‘and processing: processing of pre-mRNA: 5' cap formation, polyadenylation, splicing, + Splicing mechanisms, Splicing of tRNA precursors, Splicing of rRNA precursors. L Transcription A cell has its own language. The first step, a cell takes in reading out a needed part of its genetic instruction is to copy a particular portion of its DNA nucleotide sequence, a gene into RNA nucleotide sequence. The information in RNA, although copied into another chemical form, is still written in essentially the same language as it is in DNA. This is known as transcription. Like DNA, RNA is also a linear polymer made up of four different types of nucleotide subunits linked together by phosphodiester bonds that differ from DNA chemically in two respects: 164—_ ——_ 162_| Molecular Biology-1i wsion i, The nucleotides in RNA are ribonucleotides, i.e., they contain the sugar ribose (hence the name ribonucleic acid) rather than deoxyribose. ii, Like DNA, RNA contains the bases adenine (A), guanine (G) and cytosine (C). It contains the base uracil (U) instead of the thymine (T) in DNA. Since U, like T, can base-pair by hydrogen-bonding with A, the complementary base-pairing properties for DNA apply also to RNA (in RNA, G pairs with C and A pairs with U). It is not uncommon, however, to find other types of base pairs in RNA. For example, G pairing with U occasionally. Ribonucleotide triphosphate Direction of transcription Flap in closed Position Ribonucleotide triphosphate tunnel Active site Newly synthesized Short region of RNA transcript DNA/RNA helix Figure 1.1: RNA Transcription initiation complex There are a few significant differences, where DNA and RNA differ quite dramatically at the structural level, DNA always occurs in cells as a double-stranded helix, RNA inside the cell in transcription are single-stranded. RNA chains, therefore, fold up into a variety of shapes, just as a polypeptide chain folds up to form the final shape of a protein. The ability to fold into complex three-dimensional shapes allows some RNA molecules to have structural and catalytic functions. rH? _Ditferent Types of RNA During transcription, an enzyme system converts the genetic information in a segment of double stranded DNA into an RNA strand with a base sequence complementary to one of the DNA strands. Three major kinds of RNA are produced.Synthesis of RNA: Transcription | 163 i, Messenger RNAs (m-RNAs) encode the amino acid sequence of one or more polypeptides specified by a gene or set of genes. Transfer RNAs (t-RNAs) read the information encoded in the m-RNA and transfer the appropriate amino acid to a growing polypeptide chain during protein synthesis. iii, Ribosomal RNAs (r-RNAs) are constituents of ribosomes, the intricate cellular machines that synthesize proteins. Many additional specialized RNAs have regulatory or catalytic functions or are precursors to the three main classes of RNA. During replication, the entire chromosome is usually copied, but transcription is more selective. Only particular genes or groups of genes are transcribed at any one time, and some portions of the DNA genome are never transcribed. The cell restricts the expression of genetic information to the formation of gene products needed at any particular moment. Specific regulatory sequences mark the beginning and end of the DNA segments to be transcribed and designate which strand in duplex DNA is.to be used as the template. Similarities between DNA Replication and DNA Transcription There are few similarities observed in between DNA replication and RNA transcription. Transcription resembles replication in its fundamental chemical mechanism, (Girection of synthesis) and ts use as a template, And like replication, transcription elongation, , its polarity has initiation, and termination phases. The process of transcription has initiation which is further divided into discrete phases of DNA binding and initiation of RNA synthesis. Transcription differs from replication, in that, it does not require a primer and generally, segments of a DNA molecule. Additionally, serves as a template, involves only limited within transcribed segments, only one DNA strand i. The processes that synthesize DNA and RNA are similar in that they use similar nucleotide building blocks, They also use the same chemical met hod of attack by a terminal -OH group of the erowing chain on the triphosphate group of an incoming nucleotide. iii, Both replication and transeri iption are fueled by the hydrolysis of the Pyrophosphate group that is released upon attack,O waion 1-4] Molecular Biology! Differences between Replication and Transcription i Replication Transcription \. | DNA replication copies an entire helix. DNA transcription only transcribes specific regiong of one strand of the helix. The process is different from that in DNA ii, | replication in which the parent helix remains ‘separated until replication is done. ring DNA transcription, only short (about 60 base pairs) of the template DNA helix are unwound. As the RNA polymerase transcribes more of the DNA strand, this short stretch moves along with the transcription machinery. DNA’s nucleotides substrates are deoxyribonucleotide triphosphates. RNA’s substrates are ribonucleotide triphosphates, iv. | In DNA, the thymine base pairs with adenine. ‘Additionally, in RNA the thymine base is replaced with the base uracil DNA replication is a highly regulated process v. | that only occurs at specific times during @ cell's life. ‘DNA transcription is also regulated, but it is triggered by different signals from those used to control DNA replication. RNA primers are needed to begin replication vi. | because DNA polymerase is unable to do it alone. DNA transcription does not need the initiation of the addition of nucleotides. 2. There are two main segments of the RNA polymerase molecule: Prokaryotic RNA Polymerase i. Core enzyme and ii. Sigma subunit The main unit is together referred to as the ‘holoenzyme’. The core enzyme is itself composed of a beta, beta prime and two alpha subunits; together the core is responsible for carrying out the polymerization or synthesis of RNA. The sigma subunit of RNA polymerase is the part of the enzyme responsible for recognizing the signal on the DNA strand that tells the polymerase to begin synthesizing RNA. It is through this sigma unit that RNA polymerase is able to initiate transcription. Core enzyme: The core part can transcribe a DNA duplex after the transcription has been initiated but can't initiate transcription at proper sites, The core enzymes have the following polypeptides: a. Two a-polypeptides b. One B-polypeptideOO oa. ———————_—_} sion ‘Synthesis of RNA: Transcription | 146 c. One B'-polypeptide So the entire core enzyme is characterised as 0288". Sigma factor Core enzyme Holoenzyme Figure 1.2: RNA Polymerase ‘The a subunit: This subunit is coded by rpoA gene which is present in two copies. These components play an important role assembly of the core enzyme. It has also been reported that they play an important role in promoter recognition. ‘The B subunit: This unit has only one copy inside the core enzyme. This subunit is encoded by rpoB gene. It has an important role in binding with the incoming nucleotide to be added to the RNA chain during transcription. ‘The B subunit: This subunit is also present in one copy per molecule of core enzyme. It is coded by a gene rpoC. This subunit binds with the single stranded template DNA. Core enzyme has got the following activities, each function being specified by a specific site on it 1. DNA umwinding: Unwinding of DNA duplex structure as it moves along the DNA being transcribed. 2. Binding of antisense strand: This strand is transcribed finally. 3. Binding of sense strand: This allows antisense strand to remain single stranded. 4. DNA rewinding site: This segment helps in rewinding of DNA duplex into normal Sigma (a) Subunit:(Sigma factor is involved in the stable binding of RNA polymerase holoenzyme specifically at the promoter region. /This factor releases itself from coreMoleculer Biology. wai enzyme after the moment it reaches 10-12 nucleotides. This is the step of transcription initiation. Finally, the core enzyme continues its process of transcription. a. The E.coli s factor is composed of single polypeptide and it is encoded by the gene rpoD. Its major function is to ensure the proper binding of holoenzyme at the promoter sequences. b. If there were no s factor, then the core enzyme would bind at any other site on “DNA. So it is important to know that s factor has an important role in transcription initiation which is the recognition of promoter sites on DNA. \Also s factor does not bind DNA on its own. It needs to bind with the core enzymic. So the activity is in correlation with core enzyme. c. Sigma factor has an important role in transcription. It associates with the core enzyme and helps in promoter recognition and initiation of transcription. Finally, it separates after inion) Promoter Region \ RNA polymerase binds to specific sequences in the DNA called promoters, This directs the transcription of adjacent segments of DNA. In E, coli, RNA polymerase binding occurs within a region stretching from about 70 bp before the transcription start site to about 30 bp beyond it The DNA base pairs that correspond to the beginning of an RNA molecule are given positive numbers and those preceding the RNA start site are given negative numbery. The promoter region thus extends between positions -70 and +30. These sequences are important interaction sites for the 70 subunit. Certain nucleotides form a consensus sequence. Start Site In prokaryotic cells, free RNA polymerase molecules are constantly colliding with DNA helices. The collision leads to a weak association between the DNA and RNA polymerase, which is soon broken. However, when the RNA polymerase binds to a specific sequence on the DNA, it binds tightly, forming a DNA/RNA polymerase complex. This specific site of binding is called the start site. The start site has the following features: © The start site represents the location on the DNA that marks where the first nucleotide of an RNA chain should go; that spot is designated as the ‘plus one Position’,——________} “ ‘Synthesis of RNA: Transcription | 17 * Positions that are designated as downstream in the RNA are positively numbered according to their relative position to the plus one position. All positions designated as upstream of the start site are labeled with negative numbers according to their position relative to the start site, Sequences located just upstream of the start site, called the promoter region, contain the information that signals the RNA polymerase to start transcription. The Structure of the Promoter Region There are a number of key features to the promoter region that give it the ability to provide the signal initiating transcription. Located approximately 10 and 32 base pairs upstream of the start site are two such regions, called the -10 and ~35 sequences. Each sequence consists of six base pairs. For an ideal promoter, the sequence is TTGACA for the -35 region and TATAAT for the —10 region. 35 -10 aa pNAs'—TIGACA TATAAT 3 strand 17bp spacing Figure 1.3: Promotor region There is a third promoter element that is sometimes seen in very strong promoters which is called the UP element. It is usually composed of altemating stretches of 5 adenine and thymine bases. It is located upstream of the ~35 region. Recognition of the Promoter Region RNA polymerase binds to the DNA helix at the start site Bound to DNA, it covers a 60 base pair region within which it scans for the -35 and -10 promoters. Initially, the polymerase and specifically the sigma subunit, binds in what is called a ‘closed complex’ to the DNA. The RNA polymerase /promoter complex then undergoes a conformational change that breaks a number of base pairs extending from the —10 region to create a bubble in which the two DNA strands have separated. This bubble is usually approximately 17 base pairs in length. This new formation is called the ‘open complex’. RNA synthesis is then initiated using one of the DNA strands as a template for adding complementary RNA base pairs. Transcription is usually initiated with a purine, rather than pyrimidine, base. Once initiated, the RNA polymerase moves down the DNA strand in the elongation process, which is covered in the next section.[468] Molecular Biology-1! visi Accessory Transcription Activator Proteins CK { atove tow oki oy These proteins are required to bind to specific DNA sequences ed they help RNA polymerase “ initiate transcription via protein-protein interactions or by altering the structure of the DNA, Transcription of some promoters requires an accessory transcriptional activator; at other promoters, the activators just increase the rate of transcription but are not absolutely required. The transcription process involves three steps: i, Initiation ii, Elongation iii, Termination i. Initiation of Prokaryotic Transcription: This is one of the important phases jn transcription. This phase begins when RNA polymerase binds to the promoter as a holoenzyme and it ends when the holoenzyme Yeivey the promoter. The RNA polymerase initial binds to about 60-80 base pairs.extending from 35 to +20 sequence. This initial binding of holoenzyme to the DNA creates a closed binary complex where DNA is present as a normal double helix. ~ Te RNA synthesis] Template strand begins ‘Sigma factor Growing RNA strand ei —s ‘Termination and release of podmerase and ed RNA chain RB ‘Sigma factor Tebinds ee =: Figure 1.4: Transcription process XK Pronotev Otare aay ¢ — Syme factor ig Vuespe VA he ney { ween ‘Synthesis of RNA: Transcription | 149 Steps in Initiation Closed binary complex causes melting of DNA which is the separation of strand in a 15 bp region that includes the end of -10 sequences and extends just beyond the start point. This complex melted DNA with the holoenzyme is referred to as open binary complex which is reversible. The holoenzyme starts transcribing the antisensetrand which begins at the start point. TUmplote - The nucleotides used are ribosides 5'-triphosphate and the direction is in 5'-3" The enzymes add correct nucleotides to the deoxytibotides ofthe antisense strand. Nudlrohg After the placement of first two rit , the enzyme creates a phosphodiester bond between the two ribotides.<- ( = (4 Now these diribotides so formed remain attached with the template DNA and-the enzymes, this gives rise to a new ternary structure. € u , The holoenzyme, finally, keeps on adding the ribonucleotides to the growing RNA chain. This RNA chain remains RNA DNA hybrid at its 3' end for 2-3 nucleotides which _ is also the growing end. But 25-30 nucleotides at the growing end of the RNA chain 10. are attached with the DNA and the enzyme. Sometimes holoenzyme synthesizes a series of 2-9 bp long RNA chains which are promptly released from the ternary complex. It is referred to as the abortive‘ transcription. Elongation: Once the promoters region has been recognized by sigma factor of holoenzyme, the enzyme begins to synthesis RNA sequence and sigma factor is released. This enzyme has no exo/endo nuclease activity and cannot repair the mistakes as DNA polymerase in replication. RNA polymerase add complementary base to the template strand of DNA. It adds Thiamine for Adenine (T =A), Guanine for Cytosine (G = C), Cytosine for Guanine (C = G) and Adenine for Uracil (A = U).1210} Molecular Biology-i1 Steps in Elongation The elongation phase of transcription refers to the process through which nucleotides are added to the growing RNA chain. 1. As the RNA polymerase moves down the DNA template strand, the open complex bubble also moves. 2. The bubble is of a fixed number of nucleotides, meaning that at the leading end of the bubble, the DNA helix is being unwound, while at its trailing end, the single strands are being rejoined. 3. Whereas separation of the DNA helix_is permanent in replication, it is only temporary in transcription-and it depicts the beginning steps in transcription up to elongation and the relative positions of the bubble and the polymerase holoenzyme. Closed complex Double Polymerase ‘stranded DNA. holoenzyme | Open complex oe) * - s — oe oe obey ° Iiation | Growing RNA chain e 3 e Ng Elongation Figure 1.5: Steps in elongation‘Synthesis of RNA: Transcription | 161% As the fig./.5 shows, within the open complex bubble, the DNA and RNA form a hybrid or joint complex. The exact length of this region is unknown, but it is thought to be between 3 and 12 base pairs long and is found at the growing 3! end of the RNA. The figure also illustrates how the 5' tail end of the RNA chain is separated from, as opposed to base paited to the DNA template strand. This is’ another difference between DNA replication and\DNA transcription; in replication, the newly synthesized DNA strand remains bound in ‘helix to the strand with which it has base paired. After the inital stretch of approximately 8 base pairs has been synthesized, the sigma unit, which is responsible for recognitidn and binding 0 the promoter region, is released, The core enzyme is left to polymerize the growing RNA chain alone. This leads to the continuous extrusion of the 5' end of the.RNA from the enzyme complex. At normal room temperature, the rate of transcription in prokaryotes is 40 nucleotides per second. Termination: RNA synthesis will continue along the DNA template strand until the polymerase encounters a signal that tells\it to stop or terminate transcription. In prokaryotes, this signal can take two forms: 1. rho-independent 2. tho-dependent Rho-independent terminator The rho-independent terminator is the more simple of the two systems and as a result is also called simple termination. The rho-independent signal is found on the DNA template strand and consists of a region that contains a section that is then repeated a few base pairs away in the inverted sequence.1612 O Molecular Biology-II waion e DNA single - strand sequence a COTTAGGCTACKKXXGTAGCCTAAAAAA Transcription Hairpin loop Stem Single stranded uracils =e uw RNA sequence Figure 1.6: Rho- independent terminator As shown in the fig. 1.6, the patch is followed by a short string of adenines. When this stretch is formed transcribed into an RNA sequence, the RNA can fold back and a base pair with itself is forming a hairpin loop. The string of adenines in the DNA sequence is transcribed into uracils in the RNA sequence, Because the uracil bases will only pair weakly with the adenines, the RNA chain can easily be released from the DNA template, terminating transcription. Rho-dependent terminator (The tho-dependent terminator received its name because it is dependent on a specific Protein called a rho factof. The rho factor-is thought to bind to the end of the RNA chain and slide along the strand towards the open complex bubble, When the factor catches the polymerase, it causes the termination of transcription. The mechanism of this termination is unclear, but the rho factor could in some way pull the polymerase complex off of the DNA strand., er when “Synthesis of RA: Transcription {1043 RNA polymerase (a) Ribosome ATP Cor +P, ‘ATP ADP +P, Figure 1.7: Rho-dependent terminator 3. Transcription in Eukaryotes 3.1 Eukaryotic RNA Polymerases Unlike prokaryotic RNA polymerase that initiates the transcription of all different types of RNA, RNA polymerase in eukaryotes (including humans) comes in three variations. Each encoding a different types of gene. i. _RNA polymerase [is responsible for transeribing RNA that codes for genes that become structural components of the ribosome, a protein responsible for the translation of RNA into proteins.41614) Motecuiar Biology! vision ii. RNA polymerase II transcribes protein-encoding genes or messenger RNAS, which are the RNAs that get translated into proteins. iti/ RNA polymerase lif transcribes’a different structural region of the ribosome, transfers RNAS, which are also involved in the translation process, as well as non-protein encoding RNAs, Promoter Regions The promoter regions for RNA polymerases I and II are located upstream of the start site, but the Promoter for polymerase III is oddly located downstream. One key difference between prokaryotic and eukaryotic transcription is that eukaryotic polymerases are unable to recognize Promoter regions. They have no direct parallel to the sigma subunit of their prokaryotic ‘counterpart. Instead, eukaryotic polymerases depend on other proteins that bind to the promoter regions and then recruit the RNA polymerases to the correct spots. Unlike prokaryotic Promoters, eukaryotic promoter regions do not have a ‘canonical sequence’. They do, however, have more flexible modular elements. The polymerase II promoter, for example, has a number of traditional sequences that can appear either in tandem or alone. At the least, polymerase I] Promoters must have either a ‘TATA box’, a region approximately 25 base pairs upstream from the start-site with the sequence TATAAAA, or an ‘initiator element. The TATA box, loosely resembles the -10 region found in prokaryotic promoters, but the initiator element is not as well defined. It is known to straddle the start-site. Other non-universal sequences include the ‘CAAT box’ (GGCCAATCT) and the 'GC box' (GGGCG). Each of these sequences act as binding sites for specific transcription factors that recruits the appropriate RNA Polymerases. The most important polymerase II transcription factor is the TATA-binding protein, which binds to the TATA box promoter, where it sits on the DNA and makes contact with its minor groove. This factor is universally involved in polymerase II promoter recognition. Eukaryotic transcription uses three distinct RNA polymerases, which are specialized for different RNAs. i. RNA polymerase I makes Ribosomal RNAs, ii, _ RNA polymerase I makes messenger RNAS, iii, | RNA polymerase III makes small, stable RNAs such as transfer RNAs and 5S ribosomal RNA. Eukaryotic RNA polymerases are differentiated by their sensitivity t0 the toxic compound, a-amanitin, the active compound in the poisonous mushroom Aminita phalloides or ‘destroyingO eaten Synthesis of RNA: Transcription | 1615 angel’. RNA polymerase I is not inhibited by a-amanitin, RNA polymerase II is inhibited at very low concentrations of the drug, and RNA polymerase III is inhibited at high drug concentrations. 3.2 Mechanism of Transcription The basic mechanism of RNA synthesis by eukaryotic RNA polymerases can be divided into the following phases: iii. Initiation Elongation Termination Initiation phase: During initiation, RNA polymerase recognizes a specific site on the DNA upstream from the gene that will be transcribed called a promoter site. Most promoter sites for RNA polymerase I include a highly conserved sequence located about 25-35 bp upstream to the 5' side ofthe start site which has the consensus TATA(A/T)A(A/T) and is called the TATA box. The start site is denoted as position +1, the TATA box position is located at about position -25. The TATA box sequence resembles the -10 sequence in prokaryotes (TATAAT) except that it is located further upstream. Both elements have the same functions namely recognition by the RNA polymerase in order to'position the enzyme at the correct location to initiate transcription. TATA box also influences the efficiency of initiation. Transcription is also regulated by upstream control elements that lie 5' to the TATA box. Some eukaryotic protein-coding genes lack a TATA box and have an initiator element centered around the transcriptional initiation site. In order to initiate transcription, RNA polymerase II requires the assistance of several other proteins or protein complexes, called general or basal transcription factors which assemble into a complex on the promoter in order for RNA polymerase to bind and start transcription. These are: 1 TFIIA/Transcription Factor A 2. TFIIB/Transcription Factor B 3. TFI[D/Transcription Factor D 4. TFIIE/Transcription Factor E4616] MoeouerBioy-n SSC 5. TFIIF/Transcription Factor F 6. TFIIF/Transcription Factor H The first event in initiation is the binding of the TFIID protein complex to the TATA box via one its subunits called TBP or TATA box binding protein. TFIIA binds and stabilizes the TFIID-TATA box interaction. Next, TFIIB binds to TFIID. However, TFIIB can also bind to RNA polymerase I and so acts as a bridging protein. Thus RNA polymerase II, which has already complexed with TFIIF, now binds. This is followed by the binding of TEIE and H. This final protein complex contains at least 40 polypeptides and is called the transcription initiation complex. Promotor Transcription start site Cae TATAAA [ess -30 +1 \ RNA polymerase Il Figure 1.8: Initlation In Eukaryotes‘Synthesis of RNA: Transcription | 1647 Elongation Phase: TFIIH has two functions. They are: 1. It is a helicase which means it can use ATP to unwind the DNA helix and allows transcription to begin. 2. It phosphorylates RNA polymerase II which causes this enzyme to change its conformation and dissociate from other proteins in the initiation complex. The key phosphorylation occurs on a long C-terminal tail called the C-terminal domain (CTD) of the RNA polymerase II molecule. RNA polymerase II that has a non- phosphorylated CTD can initiate transcription but only an RNA polymerase II with a phosphorylated CTD can elongate RNA. RNA polymerase II now starts moving along the DNA template synthesizing RNA that is the process enters the elongation phase. RNA synthesis occurs in the 5' + 3! direction with the RNA polymerase catalyzing a nucleophilic attack by the 3-OH of the growing RNA chain on the alpha-phosphorus atom on an incoming ribonucleoside 5-triphosphate. The RNA molecule made from a protein- coding gene by RNA polymerase IT is called a primary transcript. Entering DNA Transcribed DNA RNA polemerase I Figure 1.9: Elongation in Eukaryotes41018 Or fp ‘Molecular Biology-1I vision Promoterclearance <— TP Tf Als Ou velege/ 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 transcript. This is called abortive imitation and is common for both eukaryotes and prokaryotes. Once the transcript reaches approximately 23 nucleotides, it no longer slips and elongation can occur, This is an ATP dependent process. TFIIH has an additional function during the initiation phase. A kinase activity in one of its subunits phosphorylates Pol II at many places in the CTD. Several other protein kinases, including CDK9 (cyclindependent kinase 9) and pTEFb (positive transcription elongation factor b) also phosphorylate the CTD, primarily on the Ser residues of the CTD repeat sequence. This Causes a conformational change in the overall complex and initiates transcription process. Phosphorylation of the CTD is also important during the subsequent elongation phase with the phosphorylation state of the CTD changing as transcription proceeds. The changes affect the interactions between the transcription complex and other proteins and enzymes such that different sets of proteins are bound at initiation thani at.later stages. During synthesis of the initial 60 to 70 nucleotides of RNA, first TFIIE and then TFIIH is released, and Pol II enters the elongation phase of transcription. Promotor 4 Coding strand See 7 5 “Template strand Figure 1.10: Promotor clearance Termination: Elongation of the RNA chain continues until termination occurs, TFIIF remains associated with Pol II throughout elongation. During this stage, the activity of the polymerase is greatly enhanced by proteins called elongation factors. The elongation factors are bound to the phosphorylated CTD suppress pausing during transcription. This also coordinate interactions between protein complexes involved in the posttranscriptional processing of mRNAs. RNA polymerase I does not terminate transcription at a specifica ——_______ ween ‘Synthesis of RNA: Transcription | 1619) site, The transcription can stop at varying distances downstream of the gene. Genes transcribed by RNA polymerase II lack any specific signals or sequences that direct RNA. Polymerase II to terminate at specific locations. RNA polymerase II can continue to transcribe RNA from a few bp to thousands of bp. The transcript is cleaved at an internal site before RNA polymerase II finishes transcribing. This cleavage site is considered the “end” of the gene. The remainder of the transcript is digested by a 5'-exonuclease called Xm2 in humans while it is still being transcribed by the RNA polymerase II. The 5'- exonulease “catches up” to RNA polymerase II by digesting away all the overhanging RNAs. Once the RNA transcript is completed, transcription is terminated. Pol II is dephosphorylated and recycled, ready to initiate another transcript. * Poly-A signal sequence Mature mRNA § Cm I agg Uncapped end of residual transcript Figure 1.14: Termination in eukaryotespg, 4-20] Molecular Borogy- whan Ribosomal RNA Synthesis Most of the RNA made in the cell is ribosomal RNA. The large and small subunit RNAs are synthesized by RNA polymerase I. Ribosomal RNA is made in a specialized organelle, the nucleolus, which contains many copies of the rRNA genes, a correspondingly large number of RNA polymerase I molecules and the cellular machinery that processes the primary transcripts into mature RNAs, RNA polymerase I is the most abundant RNA polymerase in the cell and it synthesizes RNA at the fastest rate of any of the polymerases. The genes for rRNA are present in many copies, arranged in tandem, one after the other. Each transcript contains a copy of each of three rRNAs: the 28S and 5.8S large subunit RNAs and the small subunit 18S RNA, in that order. The rRNA promoter sequences extend much further upstream than do prokaryotic promoters. The transcription of rRNA is very efficient. This is necessary because each rRNA transcript can only make one ribosome, in contrast to the large number of proteins that can be made from a single mRNA. One copy of the rRNA genes t Mogt t tesa t Cleavage sites Figure 1.12: Messenger RNA transcription The individual ribosomal RNAs must be processed from the large precursor RNA that is the product of transcription. The primary transcript contains small and large subunit RNAs in the order: 28S — 5.88 — 18S. Processing involves the modification of specific nucleotides in the IRNA, followed by cleavage of the transcript into the individual RNA components. RNA polymerase II transeribes messenger RNA and a few other small cellular RNAs. Class IlO. aa Synthesis of RNA: Transcription | 1621 promoters are usually defined by their sensitivity to a amanitin. Like prokaryotic promoters, many class II promoters contain two conserved sequences, called the CAAT and TATA boxes The TATA box is bound by a specialized transcription factor called TBP (for TATA Binding Factor). Binding of TBP is required for transcription, but other proteins are required to bind to the upstream (and potentially downstream) sequences tit are specific to each gene. Like prokaryotic transcript’, eukaryotic RNAs are initiated! with a nucleoside triphosphate. Termination of eukary: otic m-RNA transcription is |éss well understood than is termination of prokaryotic transcription, bécause the 3! ends of eukaryotic m-RNAS are derived by processing. TATA ‘Transcriptional CAAT, L start site B globin Ii Figure.1.13: CAAT and TATA box Transfer and 5S Ribosomal RNA Transcription RNA polymerase III transcribes 5S rRNA and tRNA genes. The ‘promoter’ of these transcripts can actually be located inside the gene itself, in contrast to all the other promoters. one Send of RNA RNA Control elements that 7 interact with TIA 5S gene TFIA CxS Transcription Figure 1.14: tRNA transcription1622] Molecular Biology. wision The 5' sequence is not essential for accurate transcription initiation. When the region extending from the 5' end of the gene (i.e., the part that would normally be considered to be the promoter) is deleted, RNA synthesis is carried out just as efficiently as on the native gene. The new S' end of the transcript is complementary to whatever sequences take place of the natural ones, Furthermore, initiation is only affected when sequences within the 5S rRNA gene are disrupted, The molecular explanation for this phenomenon is as follows: A protein factor binds to the 5S rRNA gene. Binding is at the internal sequence that is required for accurate initiation. The bound factor then interacts with RNA polymerase III, which is then capable of initiation. During transcription, the multiple protein factors (called TFLIs) remain bound to the transcribing gene. Post Transcriptional Modifications in Eukaryotes RNA Processing i, S' cap formation: It occurs early in transcription. Guanosyl transferase adds 5' methyl guanosine (Cap) to. 5" end of m-RNA, The cap is important for translation initiation and ‘OF export from the nucleus. One type of eukaryotic pre-mRNA modification is the addition of a structure called a 5' cap at its 5' end. At the 5' end of the mRNA, the cap consists of an additional nucleotide and methyl groups (CH) at the base of one or more nucleotides at the 5" end of the newly inserted nucleotide and the 2'-OH group of sugar. ‘After transcription initiation, the insertion of the cap takes place quickly. The 5* end of pre-mRNA. can be represented as S=pppNPNpN, in which a ribonucleotide is represented by the letter ‘N’ and a phosphate by ‘p’. One of these phosphate groups is removed shortly after the start of transcription and a guanine nucleotide is added. A special 5'-5 bond connects this guanine nucleotide to the ple-aiRNAY which “ié different from the normal 5'-3' phosphodiester bond that binds all the other RNA nucleotides, To the 5' end, ‘one or more methyl groups are added. The first of these methyl groups is attached to the position 7 of the base of the terminal guanine nucleotide making the base 7-methyl guanine. Next, in the Second anid third nucleotides, a methyl group may be attached to the 2' position of the sugar.ee Lad ‘Synthesis of RNA: Transcription RNA polymera « 8 pom Guanytransferase e () 7 NAN mm’ G-P-P-PL AS Figure 1.16: 6 mRNA capping 3 polyadenylation(A) tail: AAUAAA sequence in the RNA signals a cleavage event in the RNA. Poly(A) polymerase then adds 150-200 A residues to the 3! end of the mRNA. The poly(A) tail increases the stability of the mRNA in eukaryotes. Recent evidence has demonstrated that there are poly(A) polymerases in prokaryotes and that some m-RNAS have poly(A) tails. Interestingly though, the polyA tail destabilizes the mRNA in prokaryotes. Some a2-thalassemias (anemia due to imbalance of a and b haemoglobin subunits) have been attributed to a defect in polyadenylation. Specifically, there is a mutation in the cleavage site from AAUAAA & AAUAAG. , } Endonuctease (@) m G-P-p-P v . Cleavage site @ mM GppP NO PIV LL ) m’G-P-P-P ~ /™ pays) polymerase Figure 1.16: 3' poly(A) tailPy 1924 iii, 4. Oo. Molecular Biology-!! vision Splicing: The primary transcripts often contain intervening sequences (introns) that a removed from the RNA prior to translation by a cleavage reaction catalyzed by sn-RNp (small nuclear ribonuclear proteins which contain RNA and protein). Frequently, th, splicing site in the intron has a GU at the S' end and an AG at the 3' end. The sn-RN aligns these ends ina lariat formation to allow precise splicing, Exon 1 Intron, Exon 2 Intron exon? A exon. Splicing enzyme Figure 1.17: RNA splicing - removal of introns Splicing Mechanisms Genes in higher eukaryotes are composed of exons and introns. In the process of splicing, introns excised and exons are linked to form final mRNA. Once transcription is initiated, RNA polymerase transcribes the entire gene. The initial primary transcript produced from eukaryotic gene undergoes various processing reactions to form corresponding functional RNA\. For pre- mRNA, + 5‘ cap must be added. + 3'end must be polyadenylated. + Introns must be spliced out. + Exon ligation through splicing.a —__—_ wiION ——Syrihosis of RNA: Transcription | 1625: Mature functional RNAs are formed in nucleus and exported to cytoplasm as ribonucleoprotein component. Eukaryotic cells have RNA surveillance mechanism that prevents transport of incorrectly processed RNA to cytoplasm or they may degrade in cytoplasm if transported. RNA splicing follows cleavage and polyadenylation of 3' end of primary transcript. There are four classes of introns. Group I and Group II introns differ in their splicing mechanism. These are categorized as self-splicing introns. These were first revealed in ciliated protozoan Tetrahymena thermophila by Thomas Cesch and his colleagues. Group 1 introns are found in nuclear, mitochondrial and chloroplast gene that code for :RNA, mRNA and tRNA. Group II introns are found in primary transcript of mitochondrial or chloroplast mRNA \in fungi, algae and plants. They both do not require ATP for splicing. Splicing of exons proceed by two sequential transesterification reactions. J Group | splicing JL It requires guanosine (GMP, GDP or GTP) nucleoside or nucleotide cofactor. In the first step of splicing pathway the 3'-hydroxyl group of guanosine of intron the 3° hydroxyl of exon is displaced and acts as nucleophile. This causes precise excision of intron and ligation of exon. NO intron \ 3Intron PCE} Intermediate 5 eee UE] FEU) 2 3 SCs 9 Spliced RNA 5 eeemee{ UBT 3 Figure 1.18: Group | intron splicing mechanism Group II splicing 2-hydroxyl group of adenine residue within intron act as nucleophile. These attacks on 5' splice site to form a lariat structure. Adenosine in lariat structure has three phosphodiester bonds. The 3' hydroxyl of 5" exon acts as nucleophile.meen ‘Synthesis of RNA: Transcription | 1627 Primary A tance f rd Adenosine has throe prone tot LO ons) SIGE RNA § eee U5 eee 5 Figure 1.19: Splicing mechanism of Group Il intron Group Ill class intron These are called spliceosomal intron because their removal is catalyzed by large protein complex called spliceosomé. These are specialized RNA protein complexes also known as SnRNP or Snurp. They contain 100-200 Jong RNA known as small nuclear RNA-(SnRNA). Eukaryotic nuclei have five SARNAs U1, U2, U4, Us ‘and U6. RNA and 30 proteins in SnRNA are highly conserved. ATP is required for the formation of spliceosome. RNA cleavage-ligation nor require ATP introns have dinucleotide sequence GU at 5" end and AG at 3' end. These sequences mark splicing sites The U1 ‘SnRNA has a sequence near its 5' end complementary to splice site at 5' end of intron..U1 has pseudouridine denoted by y. UJ2 paired to intron at position encompassing A residue that acts as nucleophile. Base pairing of U2 SnRNA generates bulge that displaces and activates adenylate, The 2' end of adenylate forms lariat structure through 2',5'-phosphodiester bond. mf 7 > oy Joe oeg ‘Spliceosome formation ¢ Afier this, U4-U6 complex and US bind and form inactive spliceosome. This is converted to active spliceosome by internal rearrangement in which Ul, U4 are expelled. U6 is paired with 5'Zs 1-28} Molecular Biology-1 vision splice site and U2. Coordination of splicing and transcription brings splice site together. 4 splicing, intron in the nucleus are degraded. Ag 5 —{———————= 6 [AG es fo c 14 rt ur smn (_ (26 CH ho Ht Ang a @ 52m ( adtvatoy) ADP + P, Fvech Intron release —— 3) Figure 1.20: Splicing mechanism in mRNA.Zz ae ‘Synthesis of RNA: Transcription | 1629 | Group IV class intron \ ; Con yrnzc These are found in certain tRNA. Splicing reaction requires ATP and endonuclease ‘The endonuclease cleave phosphodiester bonds at both ends of intron. The two ends of exons are joined together by mechanism similar to DNA ligase reaction. Spliceosomal introns are limited to eukaryotes. Group I and Group II introns are found in both bacteria and bacterial viruses. Introns are found to be more common in archaea than in bacteria. Splicing of tRNA and rRNA precursor Post transcriptional processing is not limited to mRNA. Ribosomal RNA of bacterial, archael and eukaryotic cell are made from preribosomal RNA or pre-rRNA. tRNA in the same way is derived from longer precursor. These RNAs may Contain modified nucleosides. 41 Splicing of rRNA Precursors Processing in Bacteria In bacteria 16S, 23S, SS rRNA and some tRNA arise from 30S RNA precursor of 6500 nucleotides. There is removal of RNA at both ends of 30S precursor and between the segments of 30S precursor. The 16S rRNA includes pseudouridine and methylated nucleosides on base. The 23S rRNA contains 10 pseudouridine, 1 dihydrouridine and 12 methylated nucleosides. Methylation reaction requires S-adenosylmethionine. Formation of pseudouridine does not require cofactor. The segment between 16S and 23S rRNA genes encodes one or two tRNA. Different pre-rRNA transcript produces different tRNAs. The 30S RNA precursor before cleavage methylated at specific bases. Modification includes conversion of uridine to pseudouridine or dihydrouridine. Methylation reaction may occur on bases or 2'-hydroxyl group. Cleavage results formation of precursor of RNA and tRNA. RNAse P is a ribozyme. The 16S, 238 and SS rRNA result from action of specific nucleases. var voP| | 1 | 30) wowcuarBooonr SCN ae 16S tRNA 23S 5S (48) ml i‘ [own intermediates EERE) i] (ERE (Welaue 178 IRNA 258 5S eat oo | \son | Mature RNAs i?) —] imam Ct 16S rRNA IRNA 25S rRNA 5S rRNA Figure 1.21: Pre-rRNA processing In bacteria Processing in Eukaryotes 45S pre-tRNA transcripts are synthesized by RNA polymerase I. It is processed in nucleolus and forms 18S, 28S and 5.8S tRNAs. Cleavage reaction is mediated by endoribonuclease and exoribonuclease and nucleoside modification reaction. In nucleolus, there is tight coupling between rRNA transcription, rRNA maturation and ribosome assembly. The 45S precursor is methylated on base or 2'-hydroxyl group. Some uridine is converted to pseudouridine and some other modification occurs. The cleavage reaction and all modification require SnoRNAs are found in SnoRNPs in nucleolus. Ribonuclease enzymes cleave rRNA precursor and modify bases. There is a large number of small nucleolar RNAs or SnoRNAs that also guide cleavage reaction and nucleoside modification. In yeast: It involves pre-tRNA, more than 170 non-ribosomal protein, about 70 SnoRNA and 78 ribosomal protein. In human: They have greater number of modified nucleoside about 200 and SnoRNA. SSrRINA are made as separate transcript by Pol III.Corre | . Nucleolus ® SnoRNPS< | methylation. pseudouridine formation and other modifications, snoRNPs Initial cleavages of pro-rRNA @ \ - ‘Adaltones RR peed pesliaek @ exonucieoiytic. ¥ +, ee é oe 3 J J Sve pasm Mature ribosomal subunits 408 Figure 1.22: RNA Processing by Processing of pre-mRNA Synthesis of RNA: Transcription 1634P| Molecular Biology-lI vision Nucleoside Modification This involves conversion of uridine to pseudouridine and methylation at 2'-hydroxyl gn These reactions are mediated by SnoRNA-protein complexes or SnoRNP. This complex cond of a SnoRNA and four or five proteins. This includes the enzyme which is responsible q modification of bases. There are two classes of SnoRNPs that have key conserved sequen clement referred as lettered boxes. Convetre., of ° a etl, peas Liu J. — i. Box H/ACA SnoRNPs: This is involved inl pseudouridylation. RNA pairing Witir H/AC, ‘SnoRNAs guide pseudouridylations. The target RNA have pseudouridine conversion a that is paired with SnoRNA. The conserved H/ACA box sequence serves also as a prot binding site. 7 z x \ tes ANANNA ‘ACA |NNN— 0H (3) Box H Box ACA Figure 1.23: Role of SnoRNPs in pseudoridine conversion ii, Box C/D SnoRNPs: This is involved in 2'-O-methylation. RNA pairing with C/D SnoRNA guide methylation reaction. Proteins bind to highly conserved C and D box sequences. This forms larger SnoRNP.ao —_____ we ‘Synthesis of RNA: Transcription | 1633 snoRNAW Ji u) A? Box %e, / G / iy Box C’ Figure 1.24: Role of SnoRNPs in methylation The SnoRNAs are 60-300 nucleotides long. Each SnoRNA includes 10-21 nucleotide sequence that is perfectly complementary to some site on rRNA. The conserved sequence of SnoRNA fold into structure that is bound by SnoRNP proteins. 4.2 Splicing of tRNA Precursors Cells have 40 to 50 distinct tRNAs and have multiple copies of tRNA genes. Enzymatic removal of nucleotides from 5' and 3" ends from RNA precursor generates tRNAs. tRNA transcript may have intron that must be excised. Enzymatic cleavage results formation of different tRNA from single primary transcript. The endonuclease RNase P removes RNA at 5' end of tRNA. This enzyme contains protein and RNA. The RNA component is essential for its activity and also able=a os an On 4034] Molecular Biology-! vison to perform processing funtion in bacterial cell also, RNase Pacts as catalytic RNA. The ends {RNA are processed first at 5' end before 3' end. The nucleotide sequence is first removed fro: primary transcript at 5' end. ’ The 3' end of tRNA is processed by exonuclease RNase D. Posttranscriptional Processin, includes addition of 3'-terminal trinucleotide CCA(3') that has an attached amino acid, Th addition is carried out by tRNA nucleitidyltransferase. This enzyme binds three ribonucleosi, triphosphate precursor in separate active site and formas phosphodiester bond to produce CCA 3" end. The addition of CCA is not dependent on DNA or RNA template but the binding sited enzyme acts as template. The processing of tRNA. also includes modification of some bases by methylation, deamination or reduction. Pseudouridine formation includes removal of base md that reattaches to sugar through C-5. The final step of tRNA processing includes splicing of 14 nucleotide introns. Introns are present in some eukaryotic tRNAs but absent in bacterial tRNA, Figure 1.25: tRNA processingZa wsion Synthesis of RNA: Transcription | 1635, i soos Ce ! Ribose [Ribose [Ribose 4-Thiouridine (S'U) Inosine (1) 4-Methyiquanosine (mG) Uh, NH~CH,—CH=C. ° ° oO cH l 4 be , yy HN H H o* Nn o7 Ww AN 4 H [Ribose| Ribose N-lsopentenyladenosine (iA) Ribothymidine (T) Pseudourine(¥) —_Dihydrouridine (D) Figure 1.26: Modified bases of rRNA and tRNA RNA export: RNA synthesis and processing occurs in the nucleus. The mature m-RNA is then transported through.the nuclear pores in the nuclear envelope to the cytoplasm. There is a nuclear complex that is involved in the transport. This complex recognizes the 5' CAP of the m- RNA. People with systemic lupus erythematosus have antibodies directed against sn-RNP protein subunits. The significance of this is unknown.