Escherichia coli transcription termination factor Rho binds and hydrolyzes ATP using a single class of three sites

Escherichia coli transcription termination factor Rho uses the energy of ATP hydrolysis to travel 5' --> 3' along RNA. We previously showed that the hexameric Rho protein binds three molecules of ATP in active sites and that hydrolysis of the three bound ATP molecules upon RNA binding is sequential. Other models of Rho ATP hydrolysis activity have arisen from reports of additional ATP binding sites on Rho. Here we present further evidence from binding, isotope partitioning, and rapid mix/chemical quench experiments, in support of the presence of only three equivalent ATP binding sites on Rho that are catalytic sites and that fire sequentially. These results are incorporated into a proposed mechanism for directional Rho tracking along RNA.