Soaked crystals of this complex with correct or

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Unformatted text preview: anscription Kornberg and co-workers (2004) bound RNAP II to a set of synthetic oligos (partial ds DNA template and a 10 nt RNA product terminating in 3’-dA to trap polymerase in translocation state. Soaked crystals of this complex with correct or mismatched nucleotides striking differences revealed (A site and E site) Part of Rpb1 of Rpb1 Network of contacts with the GTP substrate in A site. The tripper loop of Rpb1 positions the substrate for incorporation and discriminates against improper nucleotides. of Rpb2 With the Rpb4/7 in place the clamp is forced shut. Because initiation occurs with the 12-subunit enzyme, with its clamp shut, it appears that the promoter DNA must melt before the template DNA strand can descend into the enzyme active site. It also appears that Rpb4/7 extends the dock region of the polymerase, making it easier for certain general transcription factors to bind, thereby facilitating transcription initiation OVERVIEW: RNAP II TRANSCRIPTION OVERVIEW: • • Unlike prokaryote transcription: (1) RNAP II requires general factors at Unlike general promoter for basal transcription; (2) promoters can be controlled by upstream factors, ubiquitous DNA-binding proteins that act on upstream ubiquitous promoters with consensus binding seq; (3) inducible factors activated at specific times or in specific tissues regulate promoters at response elements elements Eukaryote transcription unit: many regulatory seq found several kb upEukaryote or downstream; no defined terminators; initial transcript contains coding & noncoding regions EUKARYOTIC TRANSCRIPTION UNIT (RNAPII) EUKARYOTIC • PROMOTER: TATA box centered ~25bp upstream of PROMOTER: startpoint startpoint – Includes short elements (<10bp) that bind Includes transcription factors within ~200bp of start-site transcription – binds general transcription factors similar for all Pol II binds transcribed genes transcribed • ENHANCERS: elements up to several kb up/downstream ENHANCERS: -stimulate transcription initiation; bind specific transcription factors transcription MINIMUM REQUIREMENTS FOR RNAP II TRANSCRIPTION TRANSCRIPTION • • (i) Generic RNAP II promoter: shortest nt sequence that supports (i) RNAP II transcriptional initiation - basal transcription apparatus RNAP – Initiator (Inr): transcript start pt - no consensus seq; promoters with just Inr are recognized by RNAP II just Inr – many promoters have TATA box: ~25bp upstream from start site, only element with fixed position & found in all eu...
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