RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichia coli

Expression of virulence genes in pathogenic Escherichia coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters, but how H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA strand sequence called operon polarity suppressor (ops). RfaH–ops interactions establish long-lived RfaH–EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ(70) in the UPEC strain CFT073. We identify eight RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination, whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS–binding features, explaining minor-paralog targeting.