Interaction of ribonucleotides with T7 RNA polymerase: probable role of GTP in transcription initiation.
Interaction of ribonucleotides (NTP where N = G, A, C or U) with bacteriophage T7 RNA polymerase (T7 RNAP) was studied by fluorescence emission spectroscopy of the enzyme. From the NTP-concentration-dependent quenching of fluorescence of the enzyme, apparent dissociation constants for NTP-T7 RNAP was found to be in following order: UTP>CTP>>ATP>GTP. Acrylamide quenching of tryptophan fluorescence of free and bound enzyme suggests a conformational change, particularly in the case of GTP (and ATP). This is the first report of high affinity binding of the enzyme with purine ribonucleotides in the absence of promoter. These results also suggest that GTP may induce a promoter-specific conformation of the enzyme. The observation could account for specific requirement of GTP in transcription initiation reported earlier (1-4).
Biophysics Division, Saha Institute of Nuclear Physics, Calcutta, India.
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Sen
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Dasgupta
D017123:Bacteriophage T7 / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D012265:Ribonucleotides / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins
D012265:Ribonucleotides; D014764:Viral Proteins; D006160:Guanosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases