Interaction surface of the transcription terminator Rho required to form a complex with the C-terminal domain of the antiterminator NusG

Abstract

Rho-dependent transcription termination in bacteria requires an interaction between the terminator Rho and the antiterminator NusG. The interaction surface of the Rho–NusG complex is unknown. Here we provide direct evidence that the β-sheet bundle of the C-terminal domain of NusG (NusG-CTD) has the binding determinants for Rho, proving the hypothesis described earlier [Mooney, R. A., Schweimer, K., Rosch, P., Gottesman, M., & Landick, R., (2009). Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators. J. Mol. Biol., 391, 341–358.]. Disulfide bridges can be engineered from NusG-CTD with the surface-exposed amino acids 217 and 224 of Rho, which belong to its P-loop ATPase domain. Mutational analyses of this region of Rho revealed that a hydrophobic pocket, located behind these amino acids of Rho, is the docking site for NusG-CTD. The proximity of this region of Rho to NusG-CTD in the Rho–NusG complex was also confirmed by an efficient fluorescence resonance energy transfer between residue K224 of Rho and residue A168 of NusG-CTD. The identification of the Rho–NusG interaction surface will be useful not only in understanding the role of NusG in the termination process but also in explaining the molecular basis of the involvement of NusG-CTD in recruiting Rho and the ribosome to the same transcription machinery.