The Mg2+ requirements for rho transcription termination factor: catalysis and bicyclomycin inhibition

Kinetic studies document that the essential Escherichia coli transcription termination factor rho utilizes Mg(2+) and ATP as a substrate and requires a second Mg(2+) ion for maximum poly(C)-dependent ATP hydrolysis activity. The velocity curves show a classic nonessential Mg(2+) activation pattern in which Mg(2+) augments hydrolysis by 39% and gives a K(1)' for MgATP of 9.5 microM in the presence of excess Mg(2+) and a K(1) for MgATP of 21.2 microM under limiting Mg(2+) concentrations. Bicyclomycin (1), a commercial antibiotic that inhibits rho, weakened Mg(2+) binding at the nonessential site and disrupted the nonessential Mg(2+) activation pathway for poly(C)-dependent ATP hydrolysis. The K(i) values for 1 were 23 microM and 35 microM under excess and limiting Mg(2+) conditions, respectively, while the K(Mg(app)) for nonessential Mg(2+) increased with increasing 1 concentrations. These findings, when combined with reported mechanistic studies, provide an emerging picture of key catalytic and substrate binding sites that are necessary for rho function and that are proximal to the 1 binding site.