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A stopped‐flow kinetic analysis of substrate binding and catalysis by Escherichia coli RNA polymerase
Author(s) -
Johnson Ronald Sanders,
Strausbauch Mark
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.461.1
Subject(s) - chemistry , conformational change , pyrophosphate , reaction rate constant , elongation , dissociation constant , biophysics , nucleotide , polymerase , rna polymerase , kinetics , binding site , stereochemistry , substrate (aquarium) , escherichia coli , crystallography , biochemistry , enzyme , biology , receptor , materials science , ecology , physics , quantum mechanics , gene , metallurgy , ultimate tensile strength
To test the hypothesis that the binding of the correct NTP to a pre‐insertion site triggers rapid pyrophosphate (PP i ) 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 PP i 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 PP i release is consistent with the postulated mechanism. NSF Grant #211369