Ch_7_Transcription - DNA footrinting: Method for...

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DNA footrinting: Method for establishing where DNA binding proteins bind Important because in transcription, need polymerases, TF/enhancers, and you need to know where they bind 1. Radioactively label DNA 2. treat with DNase, cutting @ discrete sites (partial hydrolysis) Get diff sizes of cut DNA 3. Incubate with certain bainding protein, which binds to specific sequence 4. put on polyacril. Gel after protein removed (by denaturation) Separates DNA on bases of size Bound protein prevents enzyme from cutting DNA where it is bound, so you can see binding site E. Coli Transcription: Proteins bind to promoter Sigma recognizes sequence on promoter, changes shape when bound, which is sensed by transcription machinery. Transcription begins Sigma dissociates after 10-15 nucleotides are transcribed. EUKARYOTIC TRANSCRIPTION Requires transcription factors Requires TATA binding protein (TBP) Polymerase 1: rRNA(5.8S, 18S, 28S) Polymerase 2: mRNA Polymerase 3: tRNA, rRNA(5S) snRNA transcribed by Pol2 and Pol3. Polymerase 1 rRNA Transcription- Yields a 45S pre-rRNA, which is cut to 28S, 18S, and 5.8S . UBF (upstream binding factor) and SL1 (selectivity factor 1- contains TBP, although no TATA box on rRNA ) bind to promoter → Polymerase I binds and transcription begins. Polymerase 2 mRNA Transcription- TFIID binds TATA box (the TATA binding protein (TBP) of TFIID) → TFIIB binds TBP → RNA Polymerase + TFIIF bind → TFIIE and TFIIH bind. (H functions as helicase and to phosphorylate C-terminal domain, which releases the polymerase from the complex) ALSO, some RNA polymerase II promoters contain Inr(initiator) element, with no TATA box. They also have a downstream promoter element (DPE). These proteins: TFIID (the TAF subunits, not TFB b/c no TATA box) binds to the Inr and DPE sequences. → TBP then attaches to promoter → TFIIB binds → Polymerase II + TFIIF bind → TFIIE and H bind, transcription begins.
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NOTE- mediator protein complex - released from CTD of polymerase II after assembly of preinitiation complex, allowing transcriptional elongation to occur. CTD initially phosphorylated by ONE phosphate. TFIIH adds more. Phosphorylation attracts elongation factors: Y-S-P-T-S-P-S repeat (all have free hydroxyl group) CTD also interacts with mediator Polymerase 3: 5S rRNA - (NOTE: promoters are DOWNSTREAM of intiation site.) TFIIIA binds to promoter →TFIIIC binds→TFIIIB binds→Polymerase binds→Transcription begins tRNA- (NOTE: promoters are DOWNSTREAM of intiation site.) TFIIIC binds promoter (NO TFIIIA!!!) →TFIIIB binds→ Polymerase binds → transcription begins Splicing RNA’s -Contain TATA box and SNAP- (NOTE: promoters UPSTREAM.) Oct 1 → SNAP binds to the SNAP promoter→TFIIIB binds to TATA box (TBP of TFIIIB does the binding) →RNA polymerase binds, transcription begins Oct 1 is a protein that sits on a DNA sequence with 8 BP. For SNAP to bind to promoter, Oct 1 must be nearby!
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This note was uploaded on 03/19/2008 for the course MMG 409 taught by Professor Arvidson during the Spring '08 term at Michigan State University.

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Ch_7_Transcription - DNA footrinting: Method for...

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