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 Mg²⁺ and ATP as a substrate and requires a second Mg²⁺ ion for maximum poly(C)-dependent ATP hydrolysis activity. The velocity curves show a classic nonessential Mg²⁺ activation pattern in which Mg²⁺ augments hydrolysis by 39% and gives a K₁‘ for MgATP of 9.5 μM in the presence of excess Mg²⁺ and a K₁ for MgATP of 21.2 μM under limiting Mg²⁺ concentrations. Bicyclomycin (1), a commercial antibiotic that inhibits rho, weakened Mg²⁺ binding at the nonessential site and disrupted the nonessential Mg²⁺ activation pathway for poly(C)-dependent ATP hydrolysis. The K_i values for 1 were 23 μM and 35 μM under excess and limiting Mg²⁺ conditions, respectively, while the K_Mg(app) for nonessential Mg²⁺ 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.