Functionally important components of the transcription elongation complex involved in Rho-dependent termination

Bacterial transcription terminator, Rho is an RNA (Ribonucleic Acid)-dependent ATPase that terminates transcription. Several structures of pretermination complexes of the Rho-transcription elongation complex (EC) revealed a static picture of components of the EC that come close to the nascent RNA-bound Rho, where many of the residues of EC reside <= 10 & Aring; from the Rho residues. However, the in vitro-formed Rho-EC complexes do not reveal the in vivo Rho-EC dynamic interaction patterns during the termination process. Here we report synthetic defect analyses of various combinations of the mutations in RNAP beta, beta' and omega-subunits, NusA, NusG, and Rho proteins to delineate the functional network of this process. Several mutations in the beta-flap and beta'-Zn-finger and -Clamp helices domains of RNAP are synthetically defective in the presence of Rho mutants indicating functional involvement of these domains. Mutations in the NusA RNA-binding domains were synthetically defective with the Rho mutants suggesting its involvement. Our genetic analyses also revealed functional antagonisms between the omega-subunit of RNAP and the NusG-CTD (c-terminal domain) during termination. We concluded that the regions surrounding the RNA exit channel, the RNA-binding domains of NusA, the RNAP omega-subunit, and NusG-CTD constitute a functional network with Rho just before the onset of in vivo Rho-dependent termination. The regions surrounding the RNA exit channel, the RNA-binding domains of NusA, and NusG-CTD constitute a functional network during the Rho-dependent termination process with the Rho hexamer.