Transcription termination factor Rho prefers catalytically active elongation complexes for releasing RNA

RNA polymerase pauses at different DNA sequences during transcription elongation, and this pausing is associated with distinct conformational state(s) of the elongation complex (EC). Transcription termination by the termination factor Rho, an RNA- dependent molecular motor, requires pausing of the EC in the termination zone of Rho- dependent terminators. We hypothesized that the conformational state(s) of the EC associated with this pausing would influence the action of Rho. Analyses of the pausing behavior of the EC at the termination points of two well known Rho- dependent terminators revealed that Rho prefers actively transcribing complexes for termination. RNA release kinetics from stalled ECs showed that the rate of RNA release by Rho was reduced if the EC was irreversibly backtracked, if its RNA exit channel was modified by an RNA hairpin, or the bridge helix/trigger loop movement in its active site was perturbed. These defects were overcome significantly by enhancing the rate of ATP hydrolysis either by increasing the concentration of ATP or by using a Rho mutant with higher ATPase activity. We propose that the force generated from ATP hydrolysis of Rho is the key factor in dislodging the EC through its molecular motor action, and this process is facilitated when the EC is in a catalytically competent state, undergoing rapid "Brownian ratchet" motion.