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Binding of tfiid to tata box or assist binding of

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Unformatted text preview: or upstream factors, where contact can TFIID be made with a TAF; a major role of TAFs is to enable such links be • different TAFs contact different upstream factors, and this different interaction can assist either binding of TFIID to TATA box, or assist binding of other factors around TFIID-TATA box complex binding • specific example of specific transcription factor contributing to assembly of basal apparatus-studies on GAL4 and VP16 GAL4 • GAL4 & VP16 (herpes GAL4 simplex virus protein) are acidic activators acidic that recruit TFIIB that AFFINITY CHROMATOGRAPHY ASSAYS FOR TRANSCRIPTION FACTORS TRANSCRIPTION Factor TFIIB binds an affinity column containing the acidic activation domain of VP16: omitted -pass crude nuclear extract thorugh column, collect what binds (eluate) and test its ability to transcribe in presence of various components of apparatus -eluate can cause transcription even when TFIIB not added, indicating that TFIIB present in eluate -conversely, only thing missing from flowthrough (ie stuff that didn’t bind column) is TFIIB, as only TFIIB addition can restore transcription Weaver 1999 Molecular Biology Ed. 1 + Nuclear extract TFIIB VP16 VP16 TFIIB TFIIB VP16 TFIIB VP16 Eluate contains only TFIIB TFIIB VP16 VP16 TFIIB VP16 VP16 + TFIIB TFIIB Flowthrough is everything but TFIIB Transcription TFIIB is only factor that can excluded from added back factors and still get get transcription (note that TFIIA is not needed for in vitro transcription) Transcription Transcription will only work when TFIIB is added back AFFINITY CHROMATOGRAPHY ASSAYS FOR TRANSCRIPTION FACTORS TRANSCRIPTION • Transcription factor effects can be Transcription assayed by affinity chromatography based on factor binding to target site and general RNAP factors and • 2-step assay: DNA containing TATA 2-step box & UASGAL4 site coupled to box chromatography beads • Step 1: Gal4p & TFIID added & Step washed ---> Step 2: RNAP & all other TFIIs… Active transcription Active • Step 1: TFIIB & TFIID added & Step washed ---> Step 2: Gal4p + RNAP & other TFIIs… No transcription No Step 1 Step 2 – THUS, Gal4p recruits TFIIB to complex Weaver 2002 Molecular Biology Ed. 2 ACIDIC ACTIVATORS FACILITATE TFIIB BINDING TO PREINITIATION COMPLEX BINDING • (a) For transcription, Gal4p (a) must be present with...
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