ADP but not P(i) dissociation contributes to rate limitation for Escherichia coli Rho.
To define the molecular mechanism by which ATP hydrolysis powers the 5'-->3' travel of homohexameric Escherichia coli transcription termination factor Rho along RNA, rates for association and dissociation of non-RNA substrates and products were measured. Rapid mix/chemical quench and stopped-flow spectrofluorometry measurements were carried out with Rho and [gamma-(32)P]ATP, mantADP, or fluorescently tagged E. coli phosphate-binding protein. The results indicate that the P(i) off-rate is not rate limiting, but at approximately 90 s(-1), the ADP dissociation rate is comparable to the 30 s(-1) k(cat). Previous results indicate that the chemistry step of ATP hydrolysis by Rho is at least 10-fold faster than the overall catalytic cycle. The as yet unmeasured RNA dissociation step, which could be associated with a protein conformation change, might also be a rate-limiting factor.
Chen
Xin
X
Department of Biochemistry and Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Stitt
Barbara L
BL
D000244:Adenosine Diphosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D010710:Phosphates / Q000737:chemistry*; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry; D012234:Rho Factor / Q000737:chemistry*; D013053:Spectrophotometry / Q000379:methods; D014158:Transcription, Genetic
D010710:Phosphates; D012234:Rho Factor; D000244:Adenosine Diphosphate; D012313:RNA; D000255:Adenosine Triphosphate