Mechanism of transcript elongation control by RfaH

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DESCRIPTION (provided by applicant): In all domains of life, efficient synthesis of long RNAs requires accessory proteins that modify RNA polymerase into a highly processive state. Bacterial transcription factor RfaH, which belongs to a universally conserved family of NusG proteins, activates expression of horizontally-transferred operons encoding cell wall and capsule components, antibiotics, and virulence factors, all of which are subject to strong Rho-mediated polarity. RfaH action depends on a DNA sequence called ops that mediates RfaH recruitment to RNA polymerase during elongation. We carried out detailed analysis of RfaH effects on transcription and identified determinants for its interactions with RNA polymerase and the ops DNA. These studies allowed us to propose a model for RfaH interactions with the transcription elongation complex and suggested that RfaH acts as a processivity clamp which stabilizes RNA polymerase contacts with DNA and RNA. Although studies in other groups confirmed that this mechanism appears to be ancient and ubiquitous, our results revealed that it makes a relatively small contribution to a dramatic, hundred-fold activation of gene expression by RfaH. The main effect of RfaH appears to rely on blocking Rho-dependent termination by competing with a Rho cofactor NusG and recruiting the ribosome to the nascent mRNA. The second mode of action is mediated by interactions between RfaH and ribosomal protein S10, which require a complete refolding of the C-terminal domain of RfaH from an α-helical hairpin into ß-barrel. This metamorphosis is as dramatic as the one proposed to occur during formation of an infectious form of prions. In this proposal, we will use a combination of biochemical, genetic, and structural approaches to pursue these unexpected findings. First, we will study conformational transitions of RfaH C-terminal domain during its life cycle, from synthesis on the ribosome to hypothetical recycling upon dissociation from RNA polymerase at the end of an operon. Second, we will follow the events that trigger RfaH domain dissociation, a prelude to refolding, during recruitment at the ops site. Third, we will begin to elucidate the mechanism by which RfaH activates translation of mRNAs that lack functional Shine- Dalgarno elements.