504_Lecture(2-10-06)-2

504_Lecture(2-10-06)-2 - Eukaryotic Transcription I....

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Unformatted text preview: Eukaryotic Transcription I. Differences between eukaryotic versus prokaryotic transcription. II. Eukaryotic RNA polymerase (core vs holoenzyme): RNA polymerase II- Promotor elements.- General Pol II transcription factors (GTF).- TBP and TAFs.- Mediator / SRB complex.- Swi/Snf and SAGA complex. III. Regulation of RNA pol II transcription.- Transcription initiation (yeast Gal4 system)- Activators, repressor, enhancer and silencer.- Transcription elongation (HIV Tat-tar system). IV: Chromatin remodeling during transcription.- Role of Swi/Snf complex and TAFs. I. Eukaryotes vs prokaryotes transcription. Eukaryotes ! Most mRNAs are monocistronic (generally one gene for one protein). ! Transcription and translation of most eukaryotes are spatially separated. ! Multiple RNA polyermases. ! Transcription factor directly binds to the DNA template, but not to the RNA pol. ! Requires ATP for initiation. ! Nucleosome / chromatin. Prokaryotes Polycistronic mRNA. Coupling transcription and translation. Single RNA polyermase RNA pol holoenzyme itself, by associated sigma factor. No ATP Naked DNA I. Eukaryotes vs prokaryotes transcription. Eukaryotic RNA polymerase RNA polymerase II is capable of transcribing DNA templates, but it will not begin transcription at the correct site. Hence it is competent for elongation but not initiation. ? ? ? Hypothesis: There must be a cis-acting DNA sequence and trans-acting factor that recruits or direct RNA Pol II at the site of transcription initiation. ??? Start of Transcription core (-45) (+40) core promoter upstream regulatory elements enhancer (-200) Core Promoter : ! Minimal region required for basal transcription. Regulatory Elements: ! Upstream regulatory sequence (Proximal element) - usually within a few hundred bases of the promoter. ! Enhancer sequence (Distal element) that functions at a distance (even downstream or within the transcription unit) and orientation independent manner . Identifying regulatory elements:- Systematically mutate DNA and connect to a convenient reporter gene. luciferase chloramphenical acetyl transferase (CAT) -galactosidase.- Transfect DNA back into a suitable eucaryotic cell.- Measure reporter gene activity. Caveats: Cell lines are usually immortal so they carry mutations in cell cycle control. Response may be cell-type specific - specific factors or combination of factors may be absent....
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This note was uploaded on 04/17/2011 for the course BIO 102 taught by Professor Staff during the Winter '11 term at UC Davis.

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504_Lecture(2-10-06)-2 - Eukaryotic Transcription I....

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