Insight into the fidelity of Escherichia coli RNA polymerase: investigation of misincorporation during transcription elongation utilizing transient state kinetics Public Deposited

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  • March 21, 2019
  • Cunningham, Candice Kermitta
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • Concentration-dependent pre-steady state kinetics of correct nucleotide incorporation led to a proposed mechanism for transcription involving multiple conformational states of RNA polymerase (RNAP). Specifically, RNAP can exist in an unactivated state or an activated state. Transition between the two states is driven by conformational changes in RNAP following templated NTP binding to an allosteric site. Further investigation led to a structural model, where the movement of the allosteric site upon NTP binding facilitates translocation of the enzyme via a ratchet motion. In this work, I use transient state kinetics to investigate the NTP concentrationdependence of misincorporation (UMP for CMP). I demonstrate misincorporation occurs only in the activated state while a subset of complexes enters into a non-productive unactivated state. Complexes in the non-productive state are trapped by an incorrect NTP bound in the catalytic site. I demonstrate the non-productive and irreversibly bound NTP is removed from the catalytic site in the presence of the correct NTP. Combining these data with structural analyses, I present a structural model for misincorporation similar to the model for correct incorporation with several key differences. I also characterize the concentration-dependent misincorporation kinetics for delta-loop RNAP with residues R542-F545 deleted from fork loop 2, the proposed allosteric site. Deletion of the four residues enhances the fidelity of RNAP, suggesting fork loop 2 is an allosteric site responsible for the fast phase of synthesis during transcription elongation. Correct and incorrect incorporation kinetic assays using RNAP with mutations in the secondary channel demonstrate that betaD675Y (E.coli) RNAP is a low fidelity variant, significantly increasing the amount of misincorporation when initiated from the promoter. I demonstrate betaD765Y RNAP exhibits a higher fidelity from purified complexes, suggesting that the experimental procedure affects the fidelity of this variant RNAP. I also reveal a zero-order dependence on the apparent rate of misincorporation with a continual increase in the extent of misincorporation for [UTP] < 75ìM in betaD675Y RNAP. Considering recent crystal structures of RNAP II and T. Thermophilus RNAP, I posit betaD675Y affects the closing of the trigger loop over the active site, thereby changing the misincorporation kinetics of the betaD675Y RNAP.
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  • Erie, Dorothy
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  • University of North Carolina at Chapel Hill
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