Evidence for DNA bending at the T7 RNA polymerase promoter.
Phage T7 RNA polymerase is the only DNA-dependent RNA polymerase for which we have a high-resolution structure of the promoter-bound complex. Recent studies with the more complex RNA polymerases have suggested a role for DNA wrapping in the initiation of transcription. Here, circular permutation gel retardation assays provide evidence that the polymerase does indeed bend its promoter DNA. A complementary set of experiments employing differential phasing from an array of phased A-tracts provides further evidence for both intrinsic and polymerase-induced bends in the T7 RNA polymerase promoter DNA. The bend in the complex is predicted to be about 40-60 degrees and to be centered around positions -2 to +1, at the start site for transcription, while the intrinsic bend is much smaller (about 10 degrees ). These results, viewed in the light of a recent crystal structure for the complex, suggest a mechanism by which binding leads directly to bending. Bending at the start site would then facilitate the melting necessary to initiate transcription.
Ujvári
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Department of Chemistry, University of Massachusetts, Amherst, MA, 01003-4510, USA.
Martin
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CT
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008957:Models, Genetic; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011487:Protein Conformation; D014158:Transcription, Genetic / Q000235:genetics; D014764:Viral Proteins
D015342:DNA Probes; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases