Functional interaction surfaces of the bacterial transcription terminator Rho are required for the interactions with the elongation factor NusG.

Transcription terminator Rho interacts with the elongation factor NusG to induce efficient termination. By employing random mutagenesis, structure-guided site-directed mutagenesis, and phenotypic re-evaluations of our laboratory mutants, we identified several mutations in the Rho-linker region (∼135-155) and the hydrophobic pocket (200-230), in addition to those observed in the C-terminal domain (CTD) region (>250). These mutants exhibited synthetic growth defects and in vivo termination defects in the presence of the NusG-CTD mutant G146D, which is defective for Rho binding. Mutants from the Rho-CTD (I382A and I382N) were the most defective for NusG binding; however, mutants from the linker (E148R) and hydrophobic pocket (P167L) were also significantly impaired. These mutants did not respond properly to NusG in the in vitro transcription reactions. Fluorescence quenching assays showed that in presence of NusG, single-cysteine derivatives at 166C (near the linker), 202C (hydrophobic pocket), and at 325C (Rho-CTD) were buried from the surface, whereas that at 80C remained exposed to the solvent. A unique mutation of Rho, H256Y, residing near the Rho-hydrophobic patch defined the pathway of Rho-CTD to its N-terminal domain (NTD) conformational changes upon binding to NusG. We propose that NusG-CTD-Rho-CTD induces a conformational change pathway to the latter's NTD to accelerate Rho-closed complex formation upon binding to the Rho utilisation sites.