ATP binding to Rho transcription termination factor. Mutant F355W ATP-induced fluorescence quenching reveals dynamic ATP binding.

Rho transcription termination factor mutant, F355W, showed tryptophan fluorescence intensity approximately twice that of wild-type Rho at equivalent protein concentrations and underwent a decrease in relative fluorescence intensity at 350 nm when 100 microm ATP was added in the presence or absence of RNA. Titration of this fluorescence quenching with varying concentrations of ATP (0-600 microm), where Rho is shown to exist as a hexamer (400 nm Rho), revealed tight and loose ATP-binding sites. Bicyclomycin, a specific inhibitor of Rho, increased the tight ATP binding and was used to calibrate ATP-induced fluorescence quenching by using [gamma-(32)P]ATP filter binding. For the Rho mutant F355W, three tight (K(d)(1) = 3 +/- 0.3 microm) and three loose (K(d)(2) = 58 +/- 3 microm) ATP-binding sites per hexamer were seen on Scatchard analysis in the absence of bicyclomycin and poly(C). In the presence of bicyclomycin, the K(d)(1) changed from 3.0 to 1.4 microm, but K(d)(2) underwent a lesser change. The non-hydrolyzable ATP analogue, gamma-S-ATP, gave a similar profile with three tight (K(d)(1) = 0.2 microm) and three loose (K(d)(2) = 70 microm) ATP-binding sites per hexamer. Adding poly(C) to F355W did not alter the K(d)(1) or K(d)(2) for ATP or for gamma-S-ATP. ADP-induced quenching produced 5.5 loose (K(d) = 92 microm) binding sites in the absence of poly(C), and the binding became weaker (K(d) = 175 microm) in the presence of poly(C). The data suggest that in the presence of ADP Rho has six equivalent nucleotide-binding sites. When ATP was added these sites converted to three tight and three loose binding loci. We propose an alternating ATP site mechanism where ATP binding creates heterogeneity in the ATP binding in adjacent subunits, and we suggest that ATP binding to a neighboring loose site stimulates hydrolysis at a neighboring tight binding site such that all six subunits can be potential "active" sites for ATP hydrolysis. The dynamic nature of the ATP binding to Rho is discussed in the terms of the mechanism of RNA tracking driven by ATP hydrolysis.