Regulation of rho-dependent transcription termination by NusG is specific to the Escherichia coli elongation complex.

Abstract

To terminate transcription in E. coli, Rho protein binds an RNA loading site on the nascent transcript, translocates 5'--> 3' along the RNA in an ATP-driven process, and, upon reaching the transcription elongation complex, brings about RNA release. Thus, the Rho-dependent termination process can be viewed, in part, as a kinetic competition between the rate of transcript elongation by RNA polymerase (RNAP) and the rate of Rho translocation along the nascent transcript. In the context of this model, NusG, which is an essential E. coli protein, regulates Rho-dependent termination in an apparently paradoxical way, increasing the rate of transcription elongation of E. coli RNAP in the absence of Rho while also shifting the sites of Rho-dependent termination upstream on the template. Here we investigate the regulation of Rho-dependent termination by NusG. Analytical ultracentrifugation was used to establish the existence of a stable complex of NusG and Rho and to demonstrate a stoichiometry of one NusG monomer per Rho hexamer. Surface plasmon resonance was used to examine the kinetics of the formation and dissociation of the NusG-Rho complex, yielding an association rate constant (k(on)) of 2.8 (+/-0.8) x 10(5) M(-)(1) s(-)(1), a dissociation rate constant (k(off)) of 3.9 (+/-0.7) x 10(-)(3) s(-)(1), and a calculated equilibrium (dissociation) constant (K(d)) of 1.5 (+/-0.3) x 10(-)(8) M. The apparent stability of the NusG-Rho complex is insensitive to changes in salt (potassium acetate) concentration between 0.05 and 0.15 M. The translocation and transcription termination activities of Rho at saturating NusG concentrations were, however, both sensitive to salt concentration over this range, suggesting that these activities do not directly reflect the stability of the NusG-Rho complex. Rho-dependent termination could be demonstrated for transcription complexes in which E. coli RNAP had been substituted by either bacteriophage SP6 or T7 RNAP. NusG, however, was not active in transcription termination assays with either of these phage RNAPs. Thus, we conclude that NusG modulates Rho-dependent termination by interacting specifically with the RNAP of the E. coli elongation complex to render the complex more susceptible to the termination activity of Rho.