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Unformatted text preview: MCB121 Basal Transcription machinery C. Gasser 2/12/2008
Reading: Watson: p. 396-403, 776-778 (for those who have only the fifth edition of Watson, this last part is in box 16-1 of that book). Problems: Problem set on Transcription Machinery (available on Web site), MBOC problem 6-38. I. Component identification for Pol II transcription A. Mutagenesis identifies promoter elements B. Biochemical fractionation of transcription factors II. The preinitiation complex and DNA interactions A. TFIID 1) Footprinting to define binding site 2) Gel shift assays to detect DNA binding 3) TBP, DNA binding, and conformational change 4) Functions of TAFIIs B. Sequential formation of the preinitiation complex 1) TFIID 2) TFIIB (TFIIA), gel shifts to evaluate binding 3) Pol II, TFIIF 4) TFIIE, TFIIH C. Promoter clearing and elongation 1) Promoter melting 2) Pol II C-terminal domain phosphorylation
Factor TFIID TFIIB TFIIA TFIIF TFIIE TFIIH Total TBP TAFIIs # Subunits 1 10-11 1 3 2-3 2 9-10 >42 kDa 38 15 - 250 35 13-37 30, 70 34, 57 35-89 > 2 MDa Function Binds TATA box, promotes TFIIB binding Additional promoter specificity, regulation DNA-binding, promotes TFIIF/ PolII binding Displaces repressors Recruits PolII to basal apparatus Stimulates TFIIH activities Helicase, ATPase, CTD kinase MCB121 Example applications of gel shift and footprinting in study of basal apparatus assembly. From: Flores, O., et al. (1991) Proc. Natl. Acad. Sci. USA 88:9999-10003. Fig. 2. Association of RNA polymerase II with DAB complex depends on TFIIF. (A) Binding reactions to a labeled oligonucleotide (free probe) were performed with the components indicated along the top of the gel. In lanes 37, a constant amount of TFIIA, -IIB, -IID, and IIF and increasing amounts of RNA polymerase IIA (5, 10, 20, and 40 ng, respectively)were added to the binding reactions. In lanes 8 12, the same conditions as in lanes 3 7 were used, except that a constant amount of RNA polymerase IIA (40 ng) and decreasing amounts of TFIIF (4, 3, 2, 1, and 0 l, respectively) were added. Arrows indicate the locations of the free probe and the indicated combinations of factors bound to the probe. Reactions in lanes 13 15 were performed as above except that TFIID, -IIB, or IIA were omitted as indicated. (B) DNase I protection ("footprint")pattern of complexes DAB, and DAB-PolF. After 30 minute incubation of factors with the labeled DNA, the mixtures were treated with DNase I, denatured and run on a polyacrylamide gel. The DNase I protection patterns of reactions containing no protein, the DAB complex, and the DABPolF complex are shown in lanes 1, 2, and 3, respectively. ...
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This note was uploaded on 04/06/2011 for the course MCB 121 taught by Professor Gasser during the Winter '09 term at UC Davis.
- Winter '09