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UTP allosterically regulates transcription initiation from the bacteriophage T7 A1 promoter by Escherichia coli RNA polymerase

Johnson, R.S.; Chester, R.E.

FASEB Journal 15(5): A901

2001


ISSN/ISBN: 0892-6638
Accession: 035998868

At elevated concentrations, UTP inhibits the formation of pppApU from the A1 promoter as catalyzed by E. coli RNA polymerase. Analysis of the mechanism of inhibition indicates that UTP is a noncompetitive inhibitor with respect to ATP. Thus, it appears that UTP binds to an allosteric site at high concentrations and inhibits pppApU formation. As monitored in filter binding assays, UTP at high concentrations stabilizes the RNA polymerase-A1 promoter open complex against disruption by heparin. The value of the apparent Km for the binding of UTP to the open complex is 1.3(+-0.4) mM. In contrast, in the presence of ATP (2 mM), the value of the apparent Km for the binding of UTP to the open complex is 7.5(+-2.2) muM. Based on the results obtained in the abortive initiation and filter binding assays, we propose a model for transcription initiation involving two UTP binding sites; a high affinity UTP binding site corresponding to the active site and a low affinity UTP binding site corresponding to an allosteric site. From the equilibria resulting from this model, we derived an equation that relates the velocity of the reaction to the concentration of UTP. Analysis of the experimental data according to this equation and assuming that the value of the apparent KUTP.I. for the binding of UTP to the low affinity allosteric binding site is 1.3(+-0.4) mM yielded estimates of the various parameters. Vmax is equal to 0.31(+-0.09) muM pppApU/min, KUTP.H, the apparent equilibrium constant for the binding of UTP to the active site, is equal to 8.7(+-4.7) muM and beta, the fractional decrease in kcat, is equal to 0.6(+-0.2). The theoretical curve obtained by using these values provides a good fit to the experimental data.

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