Force and DNA-bound proteins bias transcription output from converging or diverging genes.

Abstract

After reaching a terminator, alternatively to RNA release and dissociation of RNA polymerase (RNAP) from the template, RNAP may remain bound and rapidly diffuse along the DNA before restarting RNA synthesis from another position. We monitored transcriptional recycling using magnetic tweezers to determine the effect of low forces assisting or opposing translocation by RNAP. Up to 50% of RNAPs slid along the DNA following transcription cycles. Force biased the direction of sliding with the velocity increasing rapidly up to 0.7 pN and much more slowly above that level. Sigma factor (σ70) likely remained bound to these sliding RNAPs, enabling recognition of a distant promoter site and additional rounds of transcription. In fact, addition of NusG, which competes with σ70 for RNAP binding, but does not promote open complex formation, diminished additional rounds of transcription. Surprisingly, if a DNA-bound protein blocked sliding to a promoter, RNAP could re-initiate transcription at the roadblock instead. This alternative restart pathway was not affected by NusG, suggesting a σ-independent pathway for RNA synthesis. In convergent or divergent arrangements of genes with no roadblock, force favors transcription of one gene with respect to the other, producing negatively correlated gene expression like that observed in vivo.