week_9_lecture_1

Basal apparatus complex first step in complex

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Unformatted text preview: karyotes… – 8 bp consensus is all A-T base pairs and is similar to bacterial -10 bp sequence sequence – TATA-less promoters lack TATA box TATA (ii) Basal transcription apparatus: (ii) – RNAPII + general factors (TFIIs) constitute the basal apparatus complex – first step in complex formation is binding of TFIID to a region extending first TFIID upstream from TATA box upstream RECOGNITION: RNA POLYMERASE II CORE PROMOTER The eukaryotic core promoter refers to the minimal set of sequence elements for accurate transcription initiation by the Pol II machinery, as measured in vitro Typically ~40-60 nt long, extending either upstream or downstream from the +1 site TFIIB recognition (BRE), TATA box, the initiator (Inr), downstream promoter elements (DCEs, DPE, and MTE) Further upsteam: other regulatory elements, UASs, enhancers, silencers, boundary elements, and insulators HERPES VIRUS TK PROMOTER STUDIED WITH LINKER SCANNING MUTAGENESIS SCANNING Weaver 2002 Molecular Biology Ed. 2 HERPES VIRUS TK PROMOTER STUDIED WITH LINKER SCANNING MUTAGENESIS SCANNING RNA POLYMERASE I PROMOTER REGION: pol I is required for the expression of just one gene, rRNA precursor. There are many copies of that gene in each cell, and it is expressed at far higher levels than any other gene: so, has its own dedicated polymerase Upsteam control element where UBF binds and core (SL1 = TBP + 3TAFs specific for RNAP I transcription) Two rRNA promoter elements (RNAP I): the core element is absolutely required for any transcription to occur RNA POLYMERASE III (transcribes “short” genes) CORE PROMOTER Transcription initiation by RNA pol II (stepwise assembly) TATA is recognized by TFIID (multi-subunit complex: TBP+13TAFs, TBP-associated factors) TBP of TFIID binds TATA (TATA-binding protein) TFIID is critical factor in promoter recognition and preinitiation complex formation TBP extensively distorts the TATA sequence: provide a recruiting platform for other TFs PRIMER EXTENSION PRIMER • • • • • • • Weaver 2002 Molecular Biology Ed. 2 Primer extension: Primer (a) RNA isolated from sample (b) complementary labeled primer (b) added added (c) primer extended to 5´-end of (c) transcript with reverse transcriptase transcriptase (d) extended labeled ssDNA run (d) on gel (e) run sequencing rxn off (e) transcribed DNA using same primer primer (f) align fragment with sequence (f) to determine start site nucleotide to THIS TECHNIQUE MAY BE ALSO THIS USED TO QUANTITATE TRANSCRIPT LEVELS - THE MORE RNA, THE...
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