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-1 per 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.
Department of Biochemistry and Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA. stitt@sgi1.fels.temple.edu
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000255:Adenosine Triphosphate