Our biochemical, genetic, and structural data show that RfaH bridges two pincers of the crab-claw RNAP and acts as a processivity clamp to enable pause-free RNA synthesis. Subsequent studies in Archaea and eukaryotes confirmed that this mechanism is common to all NusG-like proteins. Surprisingly, we found that anti-pausing activity makes only a modest contribution to RfaH cellular function. While RfaH is dispensable for growth in the lab, it is essential for virulence, conjugation, and resistance to antibiotics in E. coli, Klebsiella pneumoniae and Salmonella.
RfaH activates expression of horizontally acquired operons that encode toxins, adhesins, capsules, and secretions systems. RfaH targets are located in transcriptionally-silent regions analogous to eukaryotic “heterochromatin”. Their mRNAs lack Shine-Dalgarno elements required for ribosome recruitment and are enriched in rare codons, which impede translation and make RNA an easy target for release by Rho. We posit that RfaH corrects these defects by (i) disrupting silencing filaments formed by Nucleoid-Associated Proteins, NAPs; (ii) directly recruiting and retaining the ribosome; and (iii) excluding NusG, which is a co-factor of Rho, from RNAP.
We are investigating molecular and structural details of these mechanisms and studying RfaH-mediated gene activation in the cellular context. Our findings will provide insights into maintenance and re-structuring of bacterial heterochromatin, which has only recently come to light, and into regulation of pathogenesis. Recognizing an urgent need to identify new treatments of Gram-negative infections, we are also seeking to identify RfaH inhibitors, which would selectively target virulence but not housekeeping genes, an approach that may alleviate selective pressure for resistance.