A stopped‐flow kinetic analysis of substrate binding and catalysis by Escherichia coli RNA polymerase

Abstract

To test the hypothesis that the binding of the correct NTP to a pre‐insertion site triggers rapid pyrophosphate (PPi) release followed by entry of the NTP into the active site for incorporation, we used the intrinsic protein fluorescence of the core polymerase in well‐defined elongation complexes to monitor nucleotide binding and incorporation. The increase in fluorescence in each case could be fitted by a single exponential. Plots of the observed first order rate constants versus NTP concentration were hyperbolic. This is consistent with a mechanism involving rapid binding followed by a slow conformational change. In the case of UMP incorporation, the value of the rate constant for the conformational change was 590±60 s−1 and the value of the apparent dissociation constant for UTP was 8.6±3.8 μM; in the case of AMP incorporation, the corresponding values were 520±50 s−1 and 15.7±5.6 μM. The rate constant for PPi release in the presence of the next correct nucleotide for incorporation was reported previously to be 587±178 s−1. The coincidence between the rate constants for the conformational change and PPi release is consistent with the postulated mechanism. NSF Grant #211369