Chapter 12 Mechanisms of Transcription

Similarities and differences between transcription and DNA replication. (i) ribonucleotides, rather than deoxyribonucleotides. (ii) not need a primer. (iii) The RNA product does not remain base-paired to the template DNA strand. Synthesis of large number of transcripts from a single gene in a short time is possible. (iv) Transcription is less accurate than replication. 1 mistake in 10,000 Which part to transcribe and how extensively can be regulated

RNA POLYMERASE AND THE TRANSCRIPTION CYCLE RNA Polymerases come in different forms but share many features. (i) Bacteria have only a single RNA polymerase, while eukaryotic cells have three: (a) RNA Pol II is responsible for transcribing most genes - essentially all protein-encoding genes. (b) RNA Pol I and III are involved in transcribing RNA-encoding genes. Pol I transcribes the ribosomal RNA precursor gene, whereas Pol III transcribes tRNA genes, some small nuclear RNA genes, and the 5S rRNA gene. (ii) The bacterial RNA polymerase core enzyme alone is capable of synthesizing RNA and comprises two copies of the α subunit and one each of the β, β’, and ω subunits. The bacterial core enzyme can initiate transcription at any point on a DNA molecule. The addition of an initiation factor called σ factor forms the RNA polymerase holoenzyme that initiate only at promoters. (iii) The bacterial and yeast enzymes share an overall shape and organization. (iv) The shape of each enzyme resembles a crab claw. * Active center cleft *two Mg++ ions, one is tightly bound.

Transcription by RNA polymerase proceeds in a series of steps Initiation: A promoter-polymerase complex forms and undergoes structural changes. Bubble formation and initial synthesis. The most important control point. Elongation: The RNA polymerase unwinds the DNA in front and re-anneal it behind. It dissociates the growing RNA chain from the template as it moves along. It also performs proofreading function. Termination: The RNA polymerase stops and releases the RNA product.

Transcription initiation involves three defined steps (i) The RNA polymerase binds to a promoter to form the closed complex. (ii) The closed complex undergoes a transition to the open complex. (iii’) abortive initiation -up to 10 or so ribonucleotides (iii) promoter clearance/promoter escape -transition to elongation by forming a stable ternary complex, containing the enzyme, DNA, and RNA.

THE TRANSCRIPTION CYCLE IN BACTERIA Bacterial promoters vary in strength and sequence but have certain defining features The bacterial core polymerase can initiate transcription at any point on a DNA molecule. The addition of an initiation factor called σ factor converts the RNA polymerase into holoenzyme that initiates only at promoters. Predominant sigma factor σ70 The strength of a promoter: how many transcripts it initiates in a given time Promoters affect the binding, isomerization, and escape of the polymerase. Promoters with sequences closer to the concensus are stronger

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The σ factor mediates binding of polymerase to the promoter (i) σ70 factor can be divided into four regions: σ region 1 through σ region 4. (ii) Region 4 recognizes the -35 element. It has two helices that form a helix-turn-helix. Contacts major groove. (iii) Alpha helix in region 2 recognizes the -10 element. contains essential aromatic residues that interact with the nontemplate strand Involved in DNA melting. (iv) Alpha helix in region 3 recognizes the extended -10 ; region 1.2 binds discriminator. UP-element is recognized by αCTD (a carboxy terminal domain of the α subunit) (v) Regions 2 and 4 are separated by about 75A.

Transition to the open complex involves structural changes in RNA polymerase and in the promoter DNA Melting occurs between -11 and +3. (ii) In the case of σ70, the transition, often called isomerization, does not need energy. It is a spontaneous conformational change in the DNA-enzyme complex--essentially irreversible. (iii) 5 channels. Nucleotide entry channel (NTP uptake channel) is not shown. RNA exit channel, downstream DNA channel, nontemplate-strand (NT) channel, template-strand (T) channel. (iv) Two major structural changes in the enzyme: 1. The pincers clamp down tightly on the downstream DNA. 2. σ region 1.1 moves to open the path. (v) σ region 1.1 acts as a molecular mimic of DNA.

Transcription is initiated by RNA polymerase without the need for a primer The enzyme should hold the initiating ribonucleotide (usually A) and the second rigidly in the correct orientation Requirement for specific interactions between the enzyme and the first nucleotide explains why most transcripts start with A. The interactions require various parts of holoenzyme, including sigma.

During initial transcription, RNA polymerase remains stationary and pulls downstream DNA into itself How the active site translocates along the DNA template during abortive initiation Transient excursion model Inchworming model Scrunching model: polymerase remains stationary, unwinds downstream DNA, and pulls that DNA into itself

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Promoter escape involves breaking polymerase-promoter interaction and polymerase core-sigma interaction Only after producing a threshold length of ten or more nucleotides, polymerase escapes from the promoter. RNA should start threading into the RNA exit channel. Possible reason for abortive initiation before achieving escape Sigma region 3.2 linker mimics RNA and blocks RNA exit channel. In elongation phase, sigma binds polymerase weakly or can be lost from the core polymerase. Scrunching is reversed upon promoter escape. Scrunching is a way of storing energy and releasing upon escape to break free of the promoter and dislodge sigma from core.

The elongating polymerase is a processive machine that synthesizes and proofreads RNA Only 8-9 nt of growing chain remain base-paired to the DNA template. In pyrophosphorolytic editing, the enzyme uses its active site to catalyze the removal of an incorrectly inserted nucleotide by reincorporation of PPi. Longer hovering over mismatches than matches. In hydrolytic editing, the polymerase backtracks by one or more nucleotides and cleaves the RNA product, removing the error-containing sequence. Stimulated by Gre factors/TFIIS (elongation factors). Nus proteins promotes elongation and termination.

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RNA polymerase can become arrested and need removing Arrested Polymerase on a damaged DNA strand can block transcription. Bacterial TRCF (transcription-repair coupling factor) removes polymerase and recruits repair enzymes (transcription-coupled repair). TRCF has an ATPase activity. It translocates along the DNA and pushes polymerase forward. Allowing it to restart elongation or terminating transcription.

Transcription is terminated by signals within the RNA sequence Sequences called terminators trigger the elongating polymerase to dissociate from the DNA and release the RNA chain it has made. Rho-dependent terminators require an additional protein called Rho to induce termination. A ring-shaped protein with six identical subunits. Has an ATPase activity. the rut sites: about 40 nt, rich in C, no secondary structure. does not bind a transcript bound by ribosomes possible models for termination: pushing the enzyme forward, pulling RNA out of enzyme, or conformational chage

Transcription is terminated by signals within the RNA sequence Rho-independent terminators, also called intrinsic terminators, terminate without the involvement of other factors. A stem-loop structure (- 20 nt hairpin) + about 8 AU base pairs. Formation of hairpin causes termination by disrupting the elongation complex. How?

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TRANSCRIPTION IN EUKARYOTES Whereas bacteria require only one additional initiation factor (σ), several initiation factors, called the GTFs (general transcription factors), are required for efficient and promoter-specific initiation in eukaryotes. In vitro transcription with naked DNA templates vs. In vivo transcription with chromatin templates RNA polymerase II core promoters are made up of combinations of four different sequence elements The eukaryotic core promoter refers to the minimal set of sequence elements required for accurate transcription initiation by the Pol II machinery, as measured in vitro. TATA box, Initiator, BRE, DPE, etc are recognized by TBP, TFIID or TFIIB Other sequence elements are also required for efficient transcription in vivo (nucleosomal templates). They constitute the regulatory sequences.

RNA polymerase II forms a preinitiation complex with general transcription factors at the promoter. The preinitiation complex is the complete set of general transcription factors and polymerase, bound together at the promoter and poised for initiation. Formation of pre-initiation complex can begin at TATA element. The TATA element is recognized by the general transcriptional factor called TFIID. TBP (TATA binding protein) and TAFs (TBP associated factors) D-A-B-F/PolII-E-F Promoter melting requires ATP hydrolysis and is mediated by TFIIH (helicase activity).

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Promoter escape requires phosphorylation of the polymerase tail Abortive initiation Promoter escape/clearance requires ATP hydrolysis and phosphorylation of CTD by TFIIH CTD heptapeptide repeats (52 repeats in human) YSPTSPS PolIIO (hyperphosphorylated) for elongation vs. PolIIA (unphosphorylated) for initiation CTD kinases and CTD phosphatases CTD phosphorylation help shed most of GTFs and PolII escapes the promoter.

TBP binds to and distorts DNA using a beta sheet inserted into the minor groove. Binding specificity relies on ability to undergo a specific structural distortion Only a limited number of hydrogen bonds with bases in the minor groove Two pairs of phenylalanine intercalating between base pairs Phosphate backbone interaction with basic residues in the b sheet AT rich sequence – more readily distorted

The other general transcription factors TAFs. TBP is associated with about ten TAFs. binding DNA elements at the promoter histone-like TAFs not only in TFIID but also in the SAGA complex regulating the binding of TBP TFIIB. enters the preinitiation complex after TBP. also contacts PolII, blocks RNA-exit channel TFIIF. two-subunit factor associates with Pol II and is recruited to the promoter together with PolII, also stimulates elongation. TFIIE. TFIIE binds next, and has roles in the recruitment and regulation of TFIIH. TFIIH. 10 subunits, ATPase activity (transition to open complex, nucleotide excision repair) protein kinase activity (at ser 5 of CTD, promoter escape)

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In vivo, transcription initiation requires additional proteins, including the mediator complex High levels of transcription in vivo (with chromatin templates) require the Mediator Complex, transcriptional regulatory proteins, and nucleosome-modifying and chromatin remodeling factors. Activators help recruit polymerase to the promoter by stabilizing its binding. Interact with chromatin modifying and remodeling factors. Mediator is associated with CTD. Deletion of individual Mediator subunits affects expression of a small subset of genes. Different activators interact with different Mediator subunits.

Mediator consists of many subunits, some conserved from yeast to human. 7 subunits (out of more than 20) show sequence homology between yeast and human Srb4/Med7 is the only subunit essential for transcription of almost every gene. Deletion of one subunit affects expression of a group of genes. Low resolution structure suggests a similar shape for Mediators. Organized in modules, which can be dissociated in vitro Various forms of Mediators or purification artifacts?

A new set of factors stimulate Pol II elongation and RNA proofreading The transition to the elongation phase involves the Pol II enzyme losing most of its initiation factors - for example, the general transcription factors and Mediator. Elongation factors and factors required for RNA processing are recruited to PolII mainly by phosphorylated CTD. CTD lies adjacent to RNA exit channel and 7 times longer than the enzyme. P-TEFb (elongation factor, kinase): recruited by activators, phosphorylates serine 2 of CTD, phosphorylates/activates hSPT5, and recruits TAT-SF1 ELL family proteins suppress transient pausing of PolII. TFIIS: 1) reduces the length of time polymerase pauses, 2) stimulates the inherent RNAse activity in PolII—proofreading (hydrolytic editing), functionally similar to Gre factors

Elongating RNA polymerase must deal with histones in its path In vitro experiments revealed that chromatin greatly impedes transcription. FACT (facilitates chromatin transcription) activates transcription on chromatin templates in vitro. a heterodimer of Spt16 and SSRP1. genetically interacts with elongation factors, including TFIIS. FACT removes H2A/H2B dimer ahead of PolII and restores the dimer immediately behind the PolII.

Elongating polymerase is associated with a new set of protein factors required for various types of RNA processing Capping, splicing, polyadenylation. An overlap in proteins involved in elongation and RNA processing: The hSPT5 (elongation factor, bind to phosphoSer5) recruits 5’ capping enzymes to CTD (phosphoSer5). TAT-SF1 recruits splicing machinery to CTD (phosphoSer2). Interconnection of elongation, termination, and RNA processing 3 enzymatic steps in capping Phosphorylation of serine 5: recruits capping enzymes Dephosphorylation of serine 5: dissociation of capping enzymes Phosphorylation of serine 2: recruits RNA splicing machinery

Elongating polymerase is associated with a new set of protein factors required for various types of RNA processing Polyadenylation and Termination CPSF (cleavage and polyadenylation specificity factor) and CstF (cleavage stimulation factor) are carried by CTD, and bind to poly A signal sequence. Cleavage and polyadenylation by poly-A polymerase Transcription does not terminate immediately after cleavage and polyadenylation

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Transcription termination is linked to RNA destruction by a highly processive RNase Torpedo model of termination (preferred model) The end of the second RNA is not capped and is recognized by Rat1(yeast)/hXrn2, which is a highly processive RNase. It either pushes PolII forward or pull the remaining RNA from the enzyme. Allosteric model Conformational change reduces processivity of PolII One possibility is that transfer of 3’ processing enzymes to RNA triggers a conformational change.

RNA polymerases I and III recognize distinct promoters, using distinct sets of transcription factors, but still require TBP Pol I is required only for synthesis of the rRNA precursor. The promoter for the rRNA gene comprises two parts: the core element and the UCE (upstream control element). Recognized by SL1 and UBF

Pol III promoter are found downstream of transcription start site Pol III promoters come in various forms. The majority have the unusual feature of being located downstream of the transcription start site. Box A and Box B: tRNA promoter Box A and Box C: 5S rRNA promoter TATA element: U6 snRNA