Sequential Hydrolysis of ATP Molecules Bound in Interacting Catalytic Sites of Escherichia coli Transcription Termination Protein Rho

Escherichia coli transcription termination protein Rho, an RNA-dependent ATPase, disrupts transcription complexes, releasing RNA and allowing RNA polymerase to recycle. Homohexameric Rho binds three molecules of MgATP in a single class of catalytically competent sites. In rapid mix chemical quench experiments, when Rho saturated with ATP was mixed with RNA and the reaction was quenched after various times, hydrolysis of the three bound ATP molecules was not simultaneous. A hydrolysis burst of one molecule of ATP per hexamer occurred at >300 s−1, followed by steady-state hydrolysis at 30 s−1per hexamer. The burst also shows that a step following ATP hydrolysis is rate-limiting for overall catalysis and requires communication among the three catalytic sites during net ATP hydrolysis. The rate of hydrolysis of radiolabeled ATP when one labeled and two unlabeled ATP molecules are bound indicates a sequential pattern of hydrolysis. Positive cooperativity of catalysis occurs among the catalytic sites of Rho; when only one ATP molecule is bound per hexamer, ATP hydrolysis upon addition of RNA is 30-fold slower than when ATP is saturating. These behaviors are comparable to those of F1-type ATPases, with which Rho shares a number of structural features. Escherichia coli transcription termination protein Rho, an RNA-dependent ATPase, disrupts transcription complexes, releasing RNA and allowing RNA polymerase to recycle. Homohexameric Rho binds three molecules of MgATP in a single class of catalytically competent sites. In rapid mix chemical quench experiments, when Rho saturated with ATP was mixed with RNA and the reaction was quenched after various times, hydrolysis of the three bound ATP molecules was not simultaneous. A hydrolysis burst of one molecule of ATP per hexamer occurred at >300 s−1, followed by steady-state hydrolysis at 30 s−1per hexamer. The burst also shows that a step following ATP hydrolysis is rate-limiting for overall catalysis and requires communication among the three catalytic sites during net ATP hydrolysis. The rate of hydrolysis of radiolabeled ATP when one labeled and two unlabeled ATP molecules are bound indicates a sequential pattern of hydrolysis. Positive cooperativity of catalysis occurs among the catalytic sites of Rho; when only one ATP molecule is bound per hexamer, ATP hydrolysis upon addition of RNA is 30-fold slower than when ATP is saturating. These behaviors are comparable to those of F1-type ATPases, with which Rho shares a number of structural features. adenosine 5′-O-(γ-thio)triphosphate TNP-ADP, the 2′,3′-O-(2,4,6-trinitrophenyl) derivatives of ATP and ADP. Escherichia coli transcription termination protein Rho aids in the release of newly synthesized RNA from paused transcription complexes (reviewed in Ref. 1Richardson J.P. Greenblatt J. Neidhardt F.C. Escherichia coli and Salmonella. American Society for Microbiology, Washington, D. C.1996: 822-848Google Scholar). The homohexameric protein binds nascent RNA and, with the RNA-dependent hydrolysis of ATP, disrupts the ternary transcription complex, releasing product RNA and allowing RNA polymerase to recycle. The discovery of a 5′ → 3′ RNA-DNA helicase activity of Rho (2Brennan C.A. Dombroski A.J. Platt T. Cell. 1987; 48: 945-952Abstract Full Text PDF PubMed Scopus (186) Google Scholar) suggested that Rho might disrupt the RNA-DNA duplex of the transcription bubble. Recent studies of ternary transcription complexes (Refs. 3Nudler E. Mustaev A. Lukhtanov E. Goldfarb A. Cell. 1997; 89: 33-41Abstract Full Text Full Text PDF PubMed Google Scholar, 4Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Crossref PubMed Scopus (300) Google Scholar, 5Komissarova N. Kashlev M. J. Biol. Chem. 1997; 272: 15329-15338Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar and reviewed in Ref. 6Landick R. Cell. 1997; 88: 741-744Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) suggest that such disruption could be important in transcription termination, as could be the release of the nascent RNA just 5′ of the RNA-DNA duplex from its interactions with RNA polymerase. As described by Nudler et al. (7Nudler E. Avetissova E. Markovtsov V. Goldfarb A. Science. PubMed Scopus Google the of RNA with RNA polymerase 5′ from the RNA-DNA the and of an RNA polymerase the the of the and to the of the ternary transcription for Rho is one in which the binds to RNA polymerase and 5′ → 3′ the RNA as ATP, and releasing RNA from of the hexamer to 1997; PubMed Scopus Google Scholar). activity could release nascent RNA from RNA sites and could the for its RNA-DNA helicase of which might be in release from paused ternary transcription The that the number of ATP molecules per RNA is by Rho RNA and Rho RNA-DNA 1997; PubMed Scopus Google Scholar) Rho binds RNA of transcription termination sites. the of and for a D. Cell. Full Text PDF PubMed Scopus Google are to by bound to Rho from J.P. J. Biol. PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). The of Rho to RNA was as by three and three sites per hexamer J. Sci. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). 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