Modulation of transcription elongation via the main channel of Escherichia coli RNA polymerase Public Deposited

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  • March 21, 2019
  • Kennedy, Scott Robert
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • Conformational changes in RNA polymerase play an important role in the regulation of transcription elongation. Previous work has demonstrated that RNA polymerase can exist in an activated state and an unactivated state which synthesizes RNA fast or slow, respectively. Additionally, the distribution of elongation complexes between these two states has been shown to be regulated by an allosteric; putatively located in the main channel of the enzyme. In this work, I determined that the proposed NTP binding site is located in the main channel of RNA polymerase and modulates elongation by using the i plus 2 nucleotide. Using site directed mutagenesis, I show that the allosteric site is composed of fork loop 2. Additionally, I clearly show that there are at least two routes for NTPs to enter the catalytic site and that one of these entry routes is via the main channel. The main channel has been previously proposed; however, up to this point, there has been little experimental evidence supporting this idea. The data from my experiments, combined with structural evidence, I have put forth a model in which the allosteric NTP causes a conformational shift in RNAP that leads to rapid translocation and fast synthesis (ie. activated state). To further bolster the result that shows that fork loop 2 comprises an allosteric site, I also performed experiments on a mutant RNA polymerase in which a totally conserved Walker B motif that is in close proximity to fork loop 2 was removed via mutagenesis. My experimental results indicated that the Walker mutant has a reduced affinity for the allosteric NTP. These results, combined with the previous work on fork loop 2 mutants, strongly indicates that the main channel is heavily utilized in transcription elongation. Further studies will be needed to elucidated the details the role the main channel plays in transcription elongation.
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  • Erie, Dorothy
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  • University of North Carolina at Chapel Hill
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