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Transcription overview DNA is the template for transcription RNA chain growth is 5’ to 3’ Prokaryotic promoters Prokaryotic promoters Sigma factor stimulates tight bonding between RNA polymerase and promoter The carboxy terminal domain (CTD) of a binds to the UP element of a promoter. Overview of transcription initiation Included: E.coli RNA pol; a strong promoter; and heparin Lane 1: no DNA Lane 2 ATP only Lanes 3-7: increasing concentrations of other three NTPs s Stimulates Transcription Initiation Expt 1: Added [g32P]ATP, or [g32P] GTP to label first position of RNA (initiation), OR used [14C] ATP to label all C’s in the mRNA (elongation) and asked if s stimulated incorporation of the labels. Result: s appeared to stimulate both initiation and elongation. Expt 2: Allowed a certain amount of initiation to occur, and then added rifampicin to inhibit any further initiation. By ultracentrifugation, looked at lengths of mRNAs produced with and without s. Result: The presence/absence of s made no difference to the mRNA lengths, which would have NOT have been the case if s stimulated elongation. Conclusion: s stimulates initiation but not elongation of transcription. Re-use of s - rifampicin, core from rifampicin-resistant strain Experiment done at low ionic strength. Label is gamma 32P labelled NTPs to measure inititation only. Re-use of s - rifampicin, core from rifampicin-resistant strain Experiment done at low ionic strength. Lable is gamma 32P labelled NTPs to measure inititation only. +rifampicin, core from rifampicin-resistant strain Classic View of the s Cycle Caveat: Evidence for above model might be artifactual; due to harsh separation conditions of initiation vs. elongation versions of RNA Pol Recent evidence: s may stay associated with RNA Pol even during elongation. One model is that the s cycle is really just a shifting of position of s so that it becomes more loosely associated with RNA Pol. The Stochastic Model FRET analysis of sigma movement relative to the DNA FRET analysis of sigma movement (first three nucleotides)relative to the DNA Trailing edge Leading edge Local DNA Melting at the Promoter •RNA Pol-promoter complexes are more stable at elevated temps. suggesting that local melting occurs upon tight binding of RNA Pol to DNA •E. coli RNA Pol binding to T7 gene promoters caused a hyperchromic shift in the A260 of DNA: indicative of DNA strand separation •Gamper and Hearst expt. indicates that RNA Pol melts a short region of DNA (171 bp) form a transcription bubble that moves with the polymerase as it transcribes DNA. This exposes the template as RNA Pol moves along it. Figure 6.19 How large is the bubble? (Gamper and Hearst experiment 1.6 superhelical turns per polymerase = ca. 17 bp b subunit and phosphodiester bond formation Rifampicin inhibits transcription initiation. The b subunit of a rifampicin-resistant strain confers rifampicin resistance when added to the other subunits that come from a rifampicin sensitive strain. Therefore, b is important for initiation. Affinity labeling RNA Pol at its active site The b subunit was labeled in this experiment indicating that it is near the active site in RNA Pol where phosphodiester bonds are formed. “Walking the polymerase” b and b’ are involved in DNA-binding -electrostatic -involves catalytic site on or near b and melted DNA -weak interaction -hydrophobic interaction -involves b and b’ -strong interaction RNA product of transcription forms RNA-DNA hybrid with DNA template strand •RNA-DNA hybrid within elongation complex extends from the -1 position to -8 or -9 to the 3’ end of the nascent RNA •Processivity of transcription depends on at least 9 bp RNA-DNA hybrid Structure of Core RNA Polymerase •Looks like open crab claw: One half of claw made mainly of b and other half made primarily of b’ •Channel between two parts of the claw is presumably where DNA passes through •Three Asp residues at active site coordinate Mg2+ and are critical for catalysis Structure of RNA Polymerase Holoenzyme • Extensive interface between b and the s and b’ subunits. • may help pry open claw so that it can bind DNA. • Disordered loop of s appears to approach active site and lies in exit channel for RNA product. • Proximity to active site may mean s helps form first phosphodiester bond; could explain in part its role in initiation. • Blocking of exit channel by loop or linker of s may be why abortive transcripts are made. Structure of RNA Pol holoenzyme bound to DNA •DNA mainly bound to s subunit. •Three highly conserved aromatic amino acids of s are implicated in promoter melting. •Template strand of DNA threads through channel containing active site. Simple view of structure Rho independent (intrinsic) termination Rho-dependent termination