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Lecture 21 - RNAP - BIOL308.pdf

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BIOL 308
Bruce Wolff

Lecture 21Eukaryotic vs Prokaryotic TranscriptionTranscription and translation occur in separate compartments1Eukaryote premRNAs are subject to extensive posttranscriptional modification processing2Chromatinstructure in eukaryotes limits accessibility 3Transcription is tightly regulated Only 001 of genes in a typical eukaryotic cell are undergoing transcription at any given momentEukaryotic RNAP does not recognize binding site by itself4Needs general transcription factorsto helpEukaryotes are mostly multicellular organisms diff cellstissues5Three RNA polymerasesdifferent roles other RNAPs6RNA polymerase IV directs silencing of endogenous DNARoles of RNA polymerase IV in gene silencing Regulation of Transcription in EukaryotesControl of gene expression similar to the mechanisms used in prokaryotic cellsExpression I soften controlled at thelevel of transcription initiationsRegulation by proteinsthat recognize specific regulatory sequences and modulate binding and activity of RNAPHowever 3 different RNAPs in eukaryotesSeparation and purification of RNAPsEarly studies 1 rRNA genesrRNA genes have high GC content 60 vs 40rRNA genes are repetitive up to 20 000 copies of the genecellrRNA genes found in nucleolusEarly studies 2 RNA synthesis under various conditionsHigh ionic concentration RNA with low GC contentLow ionic concentration RNA with high GC content similarto rRNA2Mg low ionic strength most of transcription in nucleolus2Mn high ionic strength transcription throughout the nucleus conclusion more then one RNAP2One would work in nucleolus stimulated by low salts and Mg2One would work in nucleoplasm stimulated by high salts and MnEarly studies 3 Protein properties and behaviors in Ionexchange chromatographyPurification of different protein fractions extracted from the nucleusSalt concentration in elution buffer will be high in each subsequent elution different protein fractionsUse proteins from each fraction for in vitro transcriptionConclusion there arethree distinct nuclear RNA polymerasesthat transcribe three different sets of genesFollowing set of experiments different ionic strength and divalent metal ion conditions22RNAP I active at low ionic strength works with both Mgand Mn2RNAP II more active at high ionic strength works better with Mn2RNAP III active over a broad range of ionic strengths works better with MnEarly studies 4 Biochemical experimentsDiffering sensitivity to chemicalsMouse cell nuclei incubated with increasing concentrations of alphaamanitinproduct of mushroom Amanita bisporigera and transcripts submitted to electrophoresis RNAP II is the most sensitiveto alphaamanitinToxin binds to rpb1 subunit prevents RNAPII translocation Amanita RNAPII not affected Plant RNAPII less sensitive NOTES FROM LECTURES Page 1
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