Concerted transformation of a hyper-paused transcription complex and its reinforcing protein

Abstract

SUMMARYRfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an α-helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at anopssite, KOW is released and refolds into a β-barrel, which binds the ribosome. Our structures ofops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH reveal swiveled, pre-translocated pause states stabilized by anopshairpin in the non-template DNA. Autoinhibited RfaH binds and twists theopshairpin, expanding the RNA:DNA hybrid to 11 base pairs and triggering the KOW release. Once activated, RfaH hyper-stabilizes the pause, which thus requires anti-backtracking factors for escape. Our results suggest that the entire RfaH cycle is solely determined by theopsand RfaH sequences and provide insights into mechanisms of recruitment and metamorphosis of NusG homologs across all life.HIGHLIGHTS-The nontemplate DNA strand of anops-paused transcription complex forms a hairpin-Autoinhibited RfaH binds and twists theopshairpin to expand the RNA:DNA hybrid-RfaH-hairpin contacts are solely responsible for triggering RfaH activation-Upon recruitment, RfaH hyper-stabilizes the pause and promotes RNAP backtracking