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

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.