RNA Polymerase Translocation in Processive Transcription Elongation and Pausing: Dynamics, Force-Dependence, and Modulation by Sequence-Specific RNAP-DNA Interactions

Abstract

During transcription elongation, E. coli RNA polymerase (RNAP) alternates between periods of processive nucleotide addition and periods of pausing. Various mechanisms promote pausing. In particular, increased pausing at a “consensus pause element” of the form G-10Y-1G+1 (where Y-1 corresponds to the 3′ RNA end) has been described (Science 344:1042, 2014; Science 344:1285, 2014). Such pause sites have been shown to exhibit increased efficiency in the RNAP βD446A mutant; this effect can be understood from the interaction between WT RNAP βD446 and G+1, which stabilizes RNAP in a post-translocated register in the presence of a G+1, and thus decreases pausing (Science 344:1285, 2014).In this work, we use high-resolution optical tweezers to characterize the dynamics of both processive nucleotide elongation and pausing by WT and βD446A RNAP at very high spatial and temporal resolutions, under various force conditions. Our ability to resolve sub-second pausing events provide new insights into the dynamics of RNAP translocation and pausing during elongation, and their modulation by sequence-specific RNAP-DNA interactions.