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bioRxiv · 10.1101/2024.11.22.624876

Evidence for Stepwise Disruption of E. coli RNA Polymerase-{lambda}R Promoter Contacts and Bubble Collapse in Transcription Initiation

Rector, M. J.·Li, R.·Wang, H.-C.·Record, M. T.
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Abstract

In transcription initiation by E. coli RNA polymerase (RNAP), translocation of the RNA-DNA hybrid disrupts RNAP-promoter and {sigma}70-core RNAP contacts, releasing {sigma}70 and allowing RNAP to escape. Previously, to investigate whether RNAP-promoter contacts break step-by-step as the hybrid lengthens or concertedly at escape, we determined rate constants and activation energies of nucleotide-incorporation steps involving translocation at the {lambda}PR promoter. Trends in these quantities with hybrid length were inconsistent with concerted models and provided evidence for stepwise disruption of RNAP-promoter contacts and collapse of the upstream bubble (-1 to -11). Here we report kinetic m-values quantifying urea and glycine betaine (GB) effects on rate constants of individual nucleotide-incorporation steps and compare with m-values predicted from structural and model compound information. GB is predicted to favor and urea disfavor binding the initiating NTPs and trigger-helix formation in 2-mer (pppApU) synthesis, largely because of burial of phosphate oxygens in NTP binding and amide oxygens in trigger-helix formation. Consistent with these predictions, GB accelerates and urea retards steps synthesizing 3-mer and 4-mer. However, both solutes retard mid-initiation steps (5-mer to 9-mer synthesis) where -10 contacts are proposed to break, exposing DNA phosphates and allowing the upstream initiation bubble to collapse. Strikingly, urea greatly accelerates while GB retards the 11-mer synthesis step, where -35-contacts are proposed to break, {sigma}70 is released and RNAP escapes. Urea and GB kinetic m-values and activation energies of these steps are inconsistent with concerted models and support a stepwise model of contact disruption and bubble collapse in initiation.

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Rector, M. J., Li, R., Wang, H.-C., Record, M. T.. 2024-11-26. Evidence for Stepwise Disruption of E. coli RNA Polymerase-{lambda}R Promoter Contacts and Bubble Collapse in Transcription Initiation. https://doi.org/10.1101/2024.11.22.624876

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