Abstract
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.
Highlights
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 M ATP was added in the presence or absence of RNA
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 lower affinity site matched the reciprocal of the Km value of Rho for ATP [38]. These authors concluded that ATP binding may lead to conformational changes that create both high and low ATP affinity sites, where ATP hydrolysis occurs at the tight site in partial agreement with the three ATP-binding site models [34] and that the “weakly bound ATP would become strongly bound by a conformation change within the dimer.” [38] these authors suggested that ATP permanently bound to a tight site would block the reaction cycle
Summary
Positively charged amino acid residues have been identified on the inner face of the central hole of Rho that directly alters tracking and transcription termination at the secondary RNA-binding site [14]. These positive charged residues are located above (N-terminal subdomain) and below (C-terminal subdomain) the ATP hydrolysis pocket defined by the P-loop domain (residues 178 –185). The three -subunits alternate between three conformation states based on ATP binding: empty, loosely bound ADP and phosphate, and tight binding ATP (for review see Ref. 28) This triphasic reaction mechanism promotes ATP synthesis by energy input from the electrochemical potential of the membrane [29]. Unlike F1-ATP synthase, is a homohexamer and hydrolyzes ATP to drive its 5Ј to 3Ј translocation along the RNA
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