Abstract
Processivity is essential for gene expression: Escherichia coli RNA polymerase (RNAP) must transcribe 10+ kbp operons without failure, yet backtrack-induced long-lived pauses threaten premature termination. Gre factors stimulate transcript cleavage to rescue backtracked RNAP, whereas NusA and NusG, which bridge the expressome, respectively stimulate and suppress pausing. How they cooperate to secure full-length transcription is unclear. Using high-throughput magnetic tweezers, we reconstitute ops-induced pausing, showing that sequence context sets pause occupancy. RfaH, a NusG paralog recruited at ops and essential for long virulence operons, loads onto ops-paused RNAP by two pathways: one permitting immediate escape, the other requiring GreA rescue. We find that both NusG and RfaH nullify NusA's pause-stimulating effect, RfaH sustaining runs four times longer than NusG and replicating its role in vivo, where RfaH depletion leads to conjugation failing. This work provides the foundation for dissecting virulence operon expression and its therapeutic targeting.