Abstract
RfaH, an operon-specific regulator from the ubiquitous NusG/Spt5 family, activates transcription and translation of virulence genes. Gene control by RfaH requires dramatic structural rearrangements of its two domains. In autoinhibited RfaH, the a-helical C-terminal domain (CTD) masks the RNA polymerase (RNAP) binding site on the N-terminal domain (NTD). The domains separate on RfaH recruitment to the paused transcription elongation complex (TEC). The released RfaH-NTD binds RNAP whereas the RfaH-CTD refolds into a β-barrel and recruits a ribosome. RfaH recruitment to RNAP requires a 12-nucleotide ops sequence, an exemplar of a consensus pause element, in the non-template (NT) DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops is a chimeric pause element that induces RNAP pausing to facilitate RfaH binding and that establishes direct contacts with the RfaH-NTD. The crystal structure of the RfaH:ops complex reveals that ops forms a DNA hairpin that flips out a highly conserved T and that “presents” its central bases to be specifically recognized by RfaH-NTD. Molecular modeling of the ops-paused TEC and genetic evidence support the notion that the hairpin formation is required for RfaH recruitment. Our data suggest that the striking conformational plasticity augments the information content of a short NT DNA segment exposed on the RNAP surface, expanding a repertoire of regulators that control transcription in all domains of life.
Significance Statement RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to activate virulence genes. RfaH function depends on ops, a DNA element that is transiently exposed in the non-template strand in the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. The crystal structure of the RfaH:ops complex reveals that ops forms a hairpin that positions nucleobases for specific recognition by RfaH. Molecular modeling and functional evidence suggest that RNA polymerase pausing and ops hairpin formation are required for RfaH recruitment. Our findings argue that both the primary sequence and the structure are read out by transcription factors that are recruited to the non-template DNA.