Single Molecule Studies Reveal the Mechanism and Energetics of Transcriptional Termination

Abstract

Intrinsic transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a G:C‐rich RNA hairpin followed by a 7–9 nt, U‐rich tract. We used single‐molecule optical trapping techniques to investigate the mechanism by which elements from three representative terminators (his, t500, tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias DNA translocation did not affect termination efficiency (TE). However, the force‐dependent kinetics of release at the t500 terminator and the force‐dependent TE of a mutant imply a forward translocation mechanism for this terminator. Tension applied to isolated U‐tracts induced transcript release in a manner consistent with shearing of the RNA:DNA hybrid. We deduce that different mechanisms of EC dissociation, involving hypertranslocation or RNA:DNA shearing, operate at terminators with different U‐tract sequences. Tension applied to RNA at full terminators suggests that closure of the final 2–3 hairpin bases supplies the energy that destabilizes the hybrid, and that competing nascent RNA structures modulate TE by slowing terminator hairpin formation. We propose a quantitative model based on energetics that successfully predicts the behavior for all three terminators and several mutant variants.