Kinetic mechanism of GTP binding and RNA synthesis during transcription initiation by bacteriophage T7 RNA polymerase.
We have used stopped-flow and rapid chemical quench-flow methods to investigate the kinetics of the early steps during transcription initiation by bacteriophage T7 RNA polymerase. Most promoters of T7 RNA polymerase initiate with two GTPs. The kinetics of GTP binding was investigated by monitoring the fluorescence changes resulting from GTP binding to polymerase and fluorescent 2-aminopurine-containing promoter DNA complex. Scheme 1 was determined from studies of T7 Phi10 promoter at 25 degrees C, where (E.D)n represents the polymerase.DNA complex in different conformations. GTPE and GTPI represent the elongating and initiating GTP molecules incorporated at the +2 and +1 positions, respectively. Our studies show that GTP at the elongation site binds with at least 10-fold tighter affinity than the GTP at the initiation site. Two conformational changes were revealed upon GTP binding to the polymerase.2-aminopurine DNA complex. The first conformational change occurred upon GTP binding to the elongation site. This conformational change was reversible, and studies with partially melted DNA and incorrect NTPs suggested that it may represent a DNA melting and/or base pairing step. A second rate-limiting conformational change whose rate was same as the maximum rate of pppGpG synthesis occurred after two GTPs were bound. As with DNA polymerases, this rate-limiting conformational change probably occurs at each NMP incorporation event and may be involved in proper positioning of the initiation and the elongating GTPs within the polymerase active site to achieve efficient and accurate RNA synthesis.
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Department of Biochemistry, Ohio State University, Columbus, Ohio 43210, USA.Patel.85@osu.edu
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D015075:2-Aminopurine / Q000378:metabolism; D017123:Bacteriophage T7 / Q000201:enzymology*; D003090:Coliphages / Q000201:enzymology*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D011401:Promoter Regions, Genetic; D012313:RNA / Q000096:biosynthesis*; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D014764:Viral Proteins; D015075:2-Aminopurine; D012313:RNA; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases