Abstract
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.
Highlights
To achieve transcript release, Rho binds an 80-base stretch of the relevant RNA [3, 4]
At physiological ATP concentration, the ATP on-rate and Pi hydrolysis product dissociation are not rate-limiting; they are faster than kcat
RNA binding at the secondary site is thought to be more directly involved in ATP hydrolysis [32, 38] and is what is represented in Scheme 5
Summary
Wild-type Rho from E. coli was prepared as previously described [13] from strain AR120/A6 containing plasmid p39ASE [14]. As a measure of background and of the efficacy of the quench, the mixture of Rho plus [␥-32P]ATP was injected into trap solution plus trichloroacetic acid, followed by addition of poly(C) 2 s later. Following hydrolysis of the ATP by Rho, when Pi is released from the active site, MDCC-PBP traps the free Pi and exhibits a fluorescence increase. To find the off-rate of Pi from Rho, in one syringe of the stopped-flow instrument was 1.5 M Rho hexamer, 9 M MDCC-PBP, 0.02 unit/ml PNP, and 0.1 mM 7-methylguanosine in TAGME buffer; the other syringe held 200 M ATP, 0.15. The radioactivity in the filtrate was determined in a liquid scintillation counter
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