Lecture 10: Transcription II

Transcription Side notes: Top hat l Which proteins target RNA polymerase to a promoter in bacteria? • • • • DNAA Sigma factors ORC complex General transcription factors Top hat 2 Which proteins target RNA polymerase to a promoter in eukaryotes? • • • • DNAA Sigma factors ORC complex General transcription factors Initiation of transcription Once RNA polymerase is in position, the complex of polymerase and promoter is called a closed complex RNA polymerase opens up about 14 bp of duplex DNA (the transcription bubble) In pol II, the transcription bubble is opened by helicase subunits of TFIIH and requires ATP ATP is not required to open DNA in pol I or pol III, bacterial or archaea RNA polymerases The RNA polymerase bound to an open region of DNA is called the open complex RNA polymerase frequently fails to make a full-length RNA on the first a empt This leads to release of short RNAs of 2-9 nucleotides, and is called abortive initiation RNA polymerase initiates synthesis of RNA Once a transcription bobble is formed, RNA polymerase holds the template and non-template strands apart The template strand binds to RNA polymerase at the active site The non-template strand is held apart by "lid", "zipper", and "rudder" regions Ribonucleotides (NTPs) enter the active site and base pair with the template strand Addition of ribonucleotides catalyzed by RNA pol Successive nucleotides are added at the 3' end of the growing RNA molecule by nucleophilic a ack, forming a phosphodiester bond and releasing pyrophosphate (this is very similar to DNA replication) The 5' end of the growing RNA leaves the polymerase via an exit channel As in DNA polymerase, two Mg2+ ions are present at the active site and catalyze the addition of nucleotides The Mg2+ ions activate the 3' OH and stabilize a negative charge on the leaving oxygen Conformational changes allow transcription to proceed Bacterial sigma factor and eukaryotic TFIIB are involved in abortive initiation. Both proteins have a loop that extends into RNA polymerase active site region The loop is in a position to block the elongating transcript, so the 100p must be moved in order for transcription to continue Displacement of the loop is thought to help the polymerase break away from the promoter - promoter clearance During promoter clearance, RNA polymerase undergoes conformational change that associates it very stably with DNA, and loosens its grip on initiation factors Eukaryotic polymerase is phosphorylated as it converts to the elongating complex Elongating polymerase is highly processive RNA polymerase remains associated with DNA and adds hundreds or thousands of bases to the growing RNA at about 20-50 nucleotides per second The transcription bubble continues to contain 10-14 unpaired bases RNA polymerase separates downstream DNA strands, and base pairing reforms a er the RNA polymerase has passed The most recently added ribonucleotides are within the transcription bubble Pol II couples transcription & RNA processing RNA pol II makes mRNA, but this is not usually in the final active form, so it Transcription Side notes: Top hat 3 Which of the following enzymes does RNA polymerase need to move into transcriptional elongation? • • • • • Helicase Sliding Clamp Primase All of the above None of the above is called the pre-mRNA Pre-mRNA must be processed. Transcriptional elongation is coupled to mRNA processing in eukaryotes This C-terminal domain (CTD) of pol II is crucial to this function - this is the Cterminus of the Rpb 1 subunit and has many repeats of a 7 amino acid sequence The phosphorylated CTD region of the Rpb 1 subunit is involved in this processing The fi h serine in the CTD heptad repeat is phosphorylated as RNA pol II clears the promoter region The phosphorylation recruits RNA-processing enzymes, such as those that add a guanosine cap to the 5' end of the mRNA Elongation is paused to allow time for capping to occur (20 - 60 bp downstream of TSS) Capping leads to phosphorylation of the second serine in the CTD heptad repeat, which causes the polymerase to resume elongation Elongation factors control transcriptional pausing Transcriptional pausing can also occur when the RNA transcript has short complementary sequences that can form a hairpin, or by a weak DNA - RNA hybrid within the bubble Backtracking may assist in error correction The elongation complex can backtrack when there is a pause in RNA synthesis The RNA polymerase reverses direction, and the most recently made RNA protrudes from the front of the complex Transcription cleavage factors can the chop o the 3' protruding RNA by enhancing the endonuclease activity of RNA polymerase, and transcription can resume This process may have role in error correction - if mismatched bases are incorporated, the DNA-RNA duplex could be distorted and cause stalling of RNA polymerase The mismatched region would then be cleaved, allowing RNA polymerase another chance to incorporate the correct nucleotide Transcript cleavage factors Examples of transcript cleavage factors are GreA and GreB in E. coli TFIIS is the transcript cleavage factor in eukaryotes Transcript cleavage factors bind within the "funnel" region of RNA polymerase When RNA polymerase backtracks, the 3' end of the RNA extrudes through the funnel Transcript cleavage factors are thought to position a metal ion at the active site, which activates a water molecule for hydrolysis of the phosphodiester bond Histone chaperones help RNA pol proceed through chromatin Nucleosomes could potentially hinder RNA polymerase Eukaryotes use histone chaperones to remove nucleosomes ahead of RNA polymerase, and reassemble them behind the polymerase Histone chaperones includes FACT (facilitates chromatin transcription), Asf1 and Spt6 The di erent chaperones bind to di erent parts of the disassembled histone octamer Transcriptional elongation produces supercoiling Transcription Side notes: The transcription bubble moves along DNA, continually unwinding the duplex This leads to changes in supercoiling - there is an increase in positive supercoiling ahead of the polymerase and an increase in negative supercoiling behind the polymerase Changes in supercoiling could cause stalling of RNA polymerase, and the tension must be relieved, by topoisomerases In E. coli, DNA gyrase removes the positive supercoils and DNA topoisomerase I removes the negative supercoils Termination in bacteria: intrinsic & rho dependent RNA polymerase must stop transcribing at the proper place and release the transcript and DNA Terminator sequences in DNA signal RNA polymerase to cease transcription The two main classes of bacterial terminators are intrinsic and Rho-dependent Termination in bacteria: intrinsic Intrinsic, or simple, terminators end transcription in the absence of any other factors Bacterial intrinsic terminators have two main features: An inverted repeat sequence that, when transcribed, forms a stem-loop in the RNA A string of 8-10 A residues. This pairs with the transcribed poly-U in the transcription bubble (a weak, unstable structure) The weak base pairing in the transcription bubble is thought to arrest transcription and the RNA hairpin is thought to interact with RNA polymerase and help pull the RNA out of the active site A similar intrinsic mechanism exists for RNA pol III genes in eukaryotes Termination in bacteria: Rho-dependent At some terminators, DNA sequence alone is not enough for termination - other protein factors are needed In E. coli, certain genes need the Rho protein to terminate transcription (these are Rho-dependent terminators; those not requiring Rho are Rho-independent) Rho-dependent genes do not have the hairpin and poly-A structures found in intrinsic terminators Rho is an hexameric ATP-ase with a ring structure. It binds to C-rich areas of RNA (rut sites) The ring is thought to have an open structure while binding to RNA Once bound, the ring is thought to close and ATP hydrolysis drives pulling of the RNA through the ring and away from the RNA polymerase RNA pol II termination Termination of RNA pol II genes is coupled to processing of the 3' end of the mRNA Most eukaryotic mRNAs have a poly-A tail (this is polyadenylation) RNA pol II continues to transcribe a er the polyadenylation signal, but the mRNA is cleaved at the poly(A) signal and the 3' end of the mRNA is processed Allosteric model: RNA pol II transcribes through the poly(A) and 3' processing signals RNA processing proteins associate with the processing signals and the CTD Cleavage or recognition of processing proteins causes conformation changes that lead dissociation of pol II from DNA Torpedo model: Transcription Side notes: Top hat 4 What is a role played by RNA pol II CTD phosphorylation? • Recruits RNA pol II to promoter • Recruits RNA processing enzymes • Initiates transcription by opening DNA Top hat 5 Match functions with protein types: • Transcriptional Pausing Control • Transcribing through chromatin • Backtracking & error correction • Relieving supercoiling • Topoisomerases • Histone chaperones • Cleavage factors • Elongation factors A er cleavage, the RNA downstream of the poly(A) site is digested by a 5' to 3' endonuclease (Rat1) This endonuclease is the "torpedo". It continues to degrade the nascent RNA until it runs into the RNA polymerase This disrupts polymerization and causes the enzyme to dissociate from the DNA