Studies of contacts between T7 RNA polymerase and its promoter reveal features in common with multisubunit RNA polymerases.
We have used UV-laser mediated cross-linking, DNase I footprinting and KMnO4 reactivity to probe the interaction between T7 RNA polymerase (RNAP) and a consensus promoter during the early stages of transcription. In a binary complex formed in the absence of substrate on a supercoiled plasmid, direct contacts were observed on the template (T) strand at positions -17, -5, and +3 and on the nontemplate (NT) strand at position -8. These contacts lie within the DNase I cleavage footprint from positions -21 to +11 on the T strand and from positions -17 to +16 on the NT strand and straddle sites of enhanced reactivity of thymines to KMnO4 at position -3 on the T strand and position -2 on the NT strand. Use of supercoiled plasmid templates has allowed the mapping of contacts in the initiation region of the promoter in the binary complex for the first time. Upon addition of GTP, T7 RNAP enters a reiterative mode of synthesis, producing a ladder of poly(G) products. Under these conditions the downstream contact on the T strand switched from position +3 to +4 and +5 while the contact at position -17 was maintained. Under conditions in which the synthesis of transcription products is limited to 6-7 nucleotides, only the contact at position -17 on the T strand was preserved. A comparison of these results with the interaction of Escherichia coli RNA polymerase at the lac promoter reveals strong similarities in the manner in which these polymerases recognize their promoters.
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Unité de Physico-chimie des Macromolécules Biologiques, CNRS:URA 1773, Institut Pasteur, Paris, France. cplace@pasteur.fr
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McAllister
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Buckle
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D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D010777:Photochemistry; D011196:Potassium Permanganate / Q000737:chemistry; D011401:Promoter Regions, Genetic; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014764:Viral Proteins
D046911:Macromolecular Substances; D014764:Viral Proteins; D011196:Potassium Permanganate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I