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Subunits bakers yeast s cerevisiae rnap ii genes rnap

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Unformatted text preview: ation The regulatory elements in bacterial, yeast, and human genes EUKARYOTIC GENE EXPRESSION EUKARYOTIC • THREE EUKARYOTIC RNAPs: EUKARYOTIC RNAPs: >500kDa, 8-14 subunits Baker’s yeast (S. cerevisiae) RNAP II: genes RNAP encoding all subunits identified encoding core EUKARYOTIC RNAP: COEUKARYOTIC IMMUNOPRECIPITATON Weaver 2002 Molecular Biology Ed. 2 • Yeast RNAP II isolated by Yeast epitope tagging one subunit (Rpb3p) of the complex (Rpb3p) • All yeast proteins labeled All with radiolabel with • Antibody added and binds Antibody epitope - complex isolated by co-I.P. by • Bound proteins resolved on Bound gel & observed by autoradiography autoradiography EUKARYOTIC RNAP: SUBUNITS EUKARYOTIC Weaver 2002 Molecular Biology Ed. 2 • Epitope taggedRpb3p isolated in Rpb3p complex with other RNAP II subunits RNAP • Cells labeled with Cells [35S] to identify S] RNAP II proteins, and [32P]ATP to P]ATP identify phosphorylated subunits subunits • 12 subunits isolated 12 by multi-step purification (right) purification HUMAN AND YEAST RNA POLYMERASE II SUBUNITS core The crystal structure of yeast RNAP reveals a deep cleft that can accept a DNA template. The catalytic center with Mg2+ lies at the bottom of the cleft. A second Mg2+ comes in bound to the Incoming ribonucleotide The transcription bubble inside the RNAP II elongation complex If RNA-DNA hybrid were longer the rudder would be in the way RNA-DNA hybrid is 8 bp long ss downstream DNA facilitates turn Proposed translocation mechanism α- amanitin binds Yeast RNAP II • • • • The crystal structure of a transcription elongation complex involving yeast RNA pol II (lacking Rpb4/7) reveals that the clamp is closed over the RNA-DNA hybrid in the enzyme’s cleft, ensuring processivity of transcription. Three loops of the clamp – the rudder, lid, and zipper – appear to play important roles in, respectively: initiating dissociation of RNA-DNA hybrid, maintaining this dissociation, and maintaining dissociation of the template DNA. The active center of the enzyme lies at the end of pore 1, which appears to be the conduit for NTPs to enter the enzyme and for extruded RNA to exit the enzyme during backtracking A bridge helix lies adjacent to the active center, and flexing of this helix could play a role in translocation during tr...
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