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Download 1 Transcription in eukaryotes Eukaryotic RNA polymerases
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Transcription in eukaryotes Eukaryotic RNA polymerases Separation of the three nuclear polymerases RNA polymerase I – nucleolar rRNA synthesizing enzyme. RNA polymerase II - nucleoplasmic enzyme. RNA polymerase III – nucleoplasmic enzyme. 1 Roles of RNA polymerases In studies RNA polymerases were used to transcribe various genes. Purified polymerases were shown to transcribe certain genes, but not others in vitro. RNA polymerase I – makes rRNA RNA polymerase II – makes heterogeneous nuclear RNA (hnRNAs) and small nuclear RNA (snRNAs) hnRNAs – precursors of mRNAs snRNAs – needed for maturation of hnRNA to mRNA Polymerase III - makes precursors of tRNAs, 5S rRNAs and some small RNAs: U6 snRNA – participates in splicing, 7SL RNA – small RNA involved in signal recognition in the synthesis of secreted proteins, 7SK RNA – small RNA of unknown function, the adenovirus VA (virus-associated RNA), and the Epstein-Barr virus EBER2 RNA. 2 Polymerase subunits The first subunit structures for eukaryotic RNA polymerase II were reported by 2 groups. Yeast model: Genes for all subunits were cloned and sequenced. Names of subunits - Rpb1-Rpb12, genes RPB1-RPB12. RPB – RNA polymerase B (not II). NB: Similarities – each contains large subunits and a variety of smaller ones These structures resemble that of the prokaryotic core polymerases, which contain high-mass subunits and a low-mass subunits (α2). Yeast RNA polymerases have 5 common subunits 3 RNA polymerase II Problem – enzyme is very complex, difficult to say which polypeptides that co-purify with the polymerase activity are really subunits or contaminants. Two approaches to study: •Separate subunits and then reconstitute to structure and check the function But: It was not yet possible to reconstitute RNA polymerase from separate subunits •Another option: find genes for all putative subunits, mutate them and look for the function. All the genes were discovered, cloned and sequenced. They clone for 12 putative subunits of yeast polymerase II. Each of polypeptides is encoded in a single gene in the yeast genome. 4 The epitope tagging •Antibodies against epitope tag allowed immunoprecipitation of the whole polymerase •Addition of strong detergent SDS leads to separation of subunits •They can be detected by electrophoresis of denatured subunits The epitope tagging Yeast RNA Pol II 5 Human RNA pol II subunits (Table 10.2) Rpb1 - ortholog of β’, binds DNA, involved in start selection Rpb2 - ortholog of β, contains active site, involved in start site selection, elongation rate Rpb3 – ortholog of α, may function with Rpb11 Rbp4 – subcomplex with Rpb7, stress response Rpb 5 – shared with PolI, II, III, target for activators Rpb 6 - shared with PolI, II, III, functions in assembly and stability Rpb7 – Subcomplex with Rpb4 Rpb 8 - shared with PolI, II, III, oligonucleotide binding domain Rpb9 – Zinc ribbon motif involved in elongation, functions in start site selection Rpb 10- shared with PolI, II, III Rpb 11- May function with Rpb3 as ortholog of α Rpb 12- ortholog of α Response of the PolΔ4/7 to a yeast promoter Kornberg et al: transcribed a construct containing yeast CYC1 promoter and a GAL4 binding site in a cell-free extract of rpb-4 cells. They supplement ed the reactions with either 4 or 7 subunits or GAL4-VP16 factor (or both). PolIIΔ4/7 was unable to recognize promoter (lane 1), with the help of transcription factor it could manage a little transcription (lane 2) The mt polymerase supplemented with 4 and 7 subunits could recognize promoter even in the absence of transcription factor. Mt polymerase with 4/7 and TF – transcription is very active. 6 Source: Molecular Biology, Weaver, 2005 7