Escherichia coli transcription termination protein rho has three hydrolytic sites for ATP.

We have determined that 3 mol of ATP or other adenine nucleotide can bind to Escherichia coli transcription termination protein rho, in the presence or absence of the RNA cofactor that is required for activation of rho's ATPase activity. Isotope trap experiments show that the three molecules of ATP bound/rho hexamer in the absence of RNA are hydrolyzed upon addition of RNA and are therefore correctly and productively bound at active sites. These results imply that rho acts as a trimer of dimers and that either the ATPase active sites are at the interface between head-to-head protein monomers, or that ATP binding induces asymmetry among rho subunits and results in the formation of functional dimers within the hexamer. We show that ATP is efficiently hydrolyzed by rho only upon RNA binding. We have measured KD values for ATP, ADP, and Pi binding to rho and have constructed a minimal kinetic mechanism for ATP hydrolysis by the enzyme.