Lecture 5.doc

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University of Alberta
Microbiology (Biological Sciences)
Nicolas Vozza

Lecture 5 1 Transcription Elongation Role of α subunit - α CTD RNAP can recognize the upstream promoter element UP. σ recognizes core promoter element (-10, -35); What recognizes UP? Gourse et al. – mutations in a subunit led to some that made RNAP incapable of responding to the UP element DNA footprinting experiments Wildtype RNAP footprints both core and UP element A mutant RNAP footprints core promoter only Lecture 5 2 Therefore the α (C-terminal domain) is responsible for interaction with the UP element. Footprinting is a method for detecting protein-DNA interactions that can tell where the target site lies on the DNA in protein binding. A protein bound on a DNA strand protects it from attack by something that will degrade the DNA. Therefore, it leaves a “footprint” on the DNA. Steps - DNAse: end label DNA (1 strand only) Allow protein to bind DNA Treat DNA-protein complex with DNAse I under mild (low conc.) conditions  1 cut per strand Remove protein, separate DNA strands, electrophorese beside untreated DNA. DMS – chemical is smaller and reveals more subtleties; Lecture 5 3 Hydroxyl radicals are even smaller. Limited proteolysis Analysis shows αNTD and αCTD fold independently to form 2 domains tethered by a flexible linker. Clevage reveals that alpha domains are linked by 13 aa Promoters w/o UP are bound by σ only UP promoters are bound by both σ and α  Stronger RNAP binding = stronger promoter αCTD also interacts with activator proteins that bind to sites outside of the core/extended promoter. Also, αNTD plays a key role in helping organize RNAP The order of subunit assembly is: Alpha > alpha2 > alpha 2b > alpha beta beta prime Hayward et al. - Mutations of αCTD up to a certain point produced RNAP that were still able to assemble. With 94 AA’s removed, RNAP could assemble (more NTD). BUT with 153 AA’s removed, no RNAP assembly (less NTD). RNAP assembly lies in alpha NTD Point mutations in the NTD that interact with β and β’ lie on one side of the αNTD. Lecture 5 4 And this is the side that faces away from alpha CTD of the alpha subunit dimer. Lecture 5 5 β involved in phosphodiester bond formation Zillig 1970 – discovered a way to purify each individual subun
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