Transcription_machinery_Handout - MCB121 Information (2...

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Unformatted text preview: MCB121 Information (2 pages) for: Basal Transcription Machinery C. Gasser 2/4/2009 Table 1. General transcription factors necessary for Pol II transcription in vitro 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.

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