BCH5413 EXAM LATEST UPDATE

Transcription and gene regulation - prokaryote v eukaryote - ANSWER Prokaryote - no nucleosome - regulatory sequences immediately available for transcription without having to regulate accessibility and chromatin structure - no nucleus - transcription tightly coupled with translation RNA synthesis - direction - type of bond (between what two things) - incoming nucleotide determined by... - ANSWER 5' -> 3' synthesis - formation of phosphodiester bond btwn/ incoming ribonucleotide and 3' hydroxyl of nascent RNA - incoming ribonucleotide determined by DNA sequence of template strand What happens after phosphodiester bond formation? - ANSWER Release of PPi - synthesis 5' -> 3' Does RNA synthesis require a primer? - ANSWER RNA synthesis and transcription does not require primer - can initiate de novo RNA transcription - carried out by.. - what does it do - separation of two DNA strands? - ANSWER transcription carried out by RNA Pol - Pol catalyzes synthesis of nascent RNA - separation of two DNA strands creates transcription bubble Rate of transcription - ANSWER eukaryotes: 15-30 nt/sec Bacteria: 50-100 nt/sec Mechanisms of bacterial transcription initiation: RNA Pol - synthesizes what from what - in E. coli how many Pol synthesize most of the DNA - ANSWER - synthesizes RNA from one strand of dsDNA template - in E. coli a single RNA Pol synthesizes most mRNA, tRNA, rRNA Holoenzyme subunits - ANSWER 4 subunits: alpha, beta, beta', sigma - Core enzyme: alpha, beta, beta' (synthesizes RNA randomly on dsDNA templates) - sigma factor: confers promoter binding and specificty Sigma factor(s) with Pol - most E. coli promoters bound by... - allows for... - sigma is released upon.... what continues transcription elongation? - alternative sigma factors recognize... - ANSWER - most E. coli promoters bound by sigma 70 associated RNA Pol - allows correct binding and transcription initiation at specific promoters - sigma is released upon transcription initiation; core enzyme continues transcription elongation - alternative sigma factors recognize different promoters Corresponding gene to holoenzyme subunit - subunit functions - ANSWER rpoA: 2 alpha subunits (enzyme assembly, promoter recognition, binds some activators) rpoB: beta subunit (catalytic center) rpoC: Beta' subunit (catalytic center) rpoD: sigma subunit (promoter specificity) What is the use of the following factors: - sigma70 - sigma 32 - sigmaE - sigma54 - sigmaF - ANSWER - sigma70: general - sigma 32: heat shock - sigmaE: heat shock - sigma54: nitrogen - sigmaF: flagellar Prokaryotic transcription: overall steps - transcription unit is.. - during transcription.. - transcription takes place in... - ANSWER - transcription unit is a sequence of DNA transcribed into a single RNA, starting at the promoter and ending at the terminator - during transcription, the bubble is maintained within bacterial RNA Pol, which unwinds and rewinds DNA, maintains the conditions of the partner and template DNA strands, and synthesizes RNA - transcription takes place in a "bubble" in which RNA is synthesized by phase pairing with one strand of DNA in the transiently unwound region. As the bubble progresses, the DNA duplex reforms behind it, displacing the RNA in the form of a single polynucleotide chain Prokaryotic RNA Pol Structure - ANSWER - ribonucleotide triphosphate tunnel (enter for elongation) - active site - RNA exit channel *jaws in closed configuration Structure of the Transcription Elongation complex - rudder - ANSWER - contacts nascent RNA and stabilizes elongation complex *dsDNA comes into Pol, non-template strand separated from template, template used for RNA synthesis, exit of DNA and RNA out of Pol Bacterial promoters - ANSWER - sequence specific binding proteins E. coli sigma 70 recognizes what two promoters - ANSWER - For E. coli sigma 70, the two core promoters recognized are -10 box and -35 box *these are considered 'core promoter elements' UP promoter element - ANSWER - sometimes in addition to core promoter elements Promoters and Promoter complexes - promoter definition - transcription initiation definition - position +1 - synthesis of RNA in 5' -> 3' direction - ANSWER - promoter: DNA sequence that binds RNA Pol to initiate transcription - transcription initiation: synthesis of first phosphodiester bond in nascent RNA - position +1: position of nucleotide in DNA template that encodes the fist nucleotide of mRNA - synthesis 5' -> 3': nucleotides added to 3' end form ribonucleotide triphosphate precursors Typical prokaryotic promoters recognized by E. coli sigma 70 - two regions and their sequences - mutations within these regions alter... - what is important - strength of promoter determined by... - region unwound by Pol is between.... - ANSWER - -10 region (Pribnow box): TATAAT consensus sequence - -35 region: TTGACA consensus sequence - different promoters have similar but not identical -10 and -35 region sequences - mutations within these regions alter promoter strength and function - distance between -10 and -35 regions important - strength of promoter mostly determined by affinity of RNA Pol for promoter DNA sequences - region unwound by Pol appears to be between -9 and +3 (includes right end of -10 sequence and extending to just downstream of transcription initiation site) How holoenzyme recognizes the promoter regulatory regions - what recognizes -10 and -35 regions - what recognizes UP promoter - which confers sequence specific binding to promoter - what subunits are involved in catalytic synthesis of RNA - ANSWER - sigma recognizes -10 and -35 regions - for UP (AT rich) promoter region, C terminal domain of two alpha subunits recognize this *sigma confers sequence specific binding to promoter *beta and beta' subunits are involved in the catalytic synthesis of RNA Where do sequence-specific interaction with the promoter occur? - ANSWER sigma subunit - sometimes alpha subunit if UP promoter is present Sigma in holoenzyme recognizes -10 element via ____ with the non-template DNA strand of promoter - ANSWER Sigma in holoenzyme recognizes -10 element via base-specific interactions with the non-template DNA strand of promoter Free sigma subunit (does/does not) recognize single stranded non-template strand - ANSWER does not *for sigma to recognize -10 and -35 regions, non-template strand has to be in double stranded conformation DNA Footprinting to identify that alpha-subunit recognizes UP promoter element - ANSWER - purified RNA Pol and DNA sequence bound together - 5' end labeled with radioactive nucleotide (side protected by RNA Pol) - region bound by RNA Pol protected from cleavage by DNase - after DNase treatment, purify DNA, run DNA on gel - missing bands identify the binding site Stages of transcription initiation - ANSWER - holoenzyme recognizes regulatory sequence in promoter and binds forming the closed promoter complex - unwind dsDNA to form open promoter complex - transcription initiates (RNA Pol generates short abortive transcripts that are unstable and released) - at 10nt in length, the complex is stable and turns from initiation to elongation Cycling of sigma during transcription - ANSWER - as Pol transitions to elongation, sigma factor is released - transcription proceeds with just core subunits until termination - at termination, RNA Pol released - nascent RNA released - released core enzyme recycles back to bind with sigma to form the holoenzyme and repeats Abortive transcription and initiation: RNA Pol at promoter - ANSWER 3 ideas: 1. movement of transient excursion: RNA Pol makes short movement along DNA to make abortive transcripts, then goes back to its original position 2. inch worming: back of RNA Pol stays stationary, as abortive transcripts are made, front edge of RNA Pol stretches/inch worms, as abortive transcript is released front of RNA Pol goes back to initial position 3. scrunching: RNA Pol doesnt move but DNA compresses/scrunches in promoter How scientists determined which theory about RNA Pol and abortive transcription is true - ANSWER FRET - donor and acceptor used; donor can be stimulated and release energy which is transferred to acceptor and will fluoresce at different wavelength - efficiency determined by proximity between donor and acceptor How FRET would work concerning RNA Pol and abortive transcription - ANSWER - linked acceptor to DNA template and donor to back end of sigma subunit - assembled open promoter complex - GTP and UTP added to start synthesis - measured degree of energy transfer between donor and acceptor (proximity) What FRET would show for: - transient excursion - inchworming - scrunching - ANSWER - transient excursion: as Pol moves forward, FRET will drop - inchworming: no change (no net separation) - scrunching: no change (no net separation) What FRET showed - ANSWER - no change; eliminated transient excursion model How to distinguish between scrunching and inchworming - what inchworming would show - results - ANSWER - acceptor on DNA and donor at front of sigma subunit *if inchworming, FRET would decrease results: no change in FRET How they directly determined scrunching was the correct model - what FRET would show - results - ANSWER link donor and acceptor on DNA - if scrunching occurs, FRET would increase results: showed increase in FRET