ATP Binding to Rho Transcription Termination Factor

MUTANT F355W ATP-INDUCED FLUORESCENCE QUENCHING REVEALS DYNAMIC ATP BINDING*

Yi XuDagger , Jerry JohnsonDagger , Harold Kohn§, and William R. WidgerDagger

From the Dagger  Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001 and the § Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360

Received for publication, December 19, 2002, and in revised form, January 22, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 µM ATP was added in the presence or absence of RNA. Titration of this fluorescence quenching with varying concentrations of ATP (0-600 µM), 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 -32P]ATP filter binding. For the Rho mutant F355W, three tight (Kd1 = 3 ± 0.3 µM) and three loose (Kd2 = 58 ± 3 µM) ATP-binding sites per hexamer were seen on Scatchard analysis in the absence of bicyclomycin and poly(C). In the presence of bicyclomycin, the Kd1 changed from 3.0 to 1.4 µM, but Kd2 underwent a lesser change. The non-hydrolyzable ATP analogue, gamma -S-ATP, gave a similar profile with three tight (Kd1 = 0.2 µM) and three loose (Kd2 = 70 µM) ATP-binding sites per hexamer. Adding poly(C) to F355W did not alter the Kd1 or Kd2 for ATP or for gamma -S-ATP. ADP-induced quenching produced 5.5 loose (Kd = 92 µM) binding sites in the absence of poly(C), and the binding became weaker (Kd = 175 µM) 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rho transcription termination factor from Escherichia coli is a homohexamer consisting of six identical 47-kDa subunits (1) arranged in a toroid structure (2, 3). Details of the reaction mechanism have been presented in several reviews (4, 5). To summarize, Rho recognizes RNA transcripts destined for Rho termination by binding to nascent RNA at poorly defined rut sites (Rho utilization) (6). Rho bound to RNA translocates 5' to 3' along the RNA driven by ATP hydrolysis (4, 7). When Rho approaches the stalled RNA polymerase, it competes with the polymerase for RNA leading to transcription termination (5, 8). The primary RNA-binding site that recognizes the rut sequences is located in the first 151 amino acid residues (9), which includes residues 61-66, GFGFLR, a conserved RNA-binding locus (10, 11).

Movement of Rho along the RNA has been postulated to occur through the central hole of the toroid ring at a secondary RNA-binding site that is distinct from the rut recognition site (12). Mutations in the Q-loop region of Rho (residues 285-292) affect secondary RNA binding and block transcription termination (13). Specific, 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). ATP hydrolysis is dependent on RNA binding at a secondary site (15, 16), and in the absence of RNA, Rho does not hydrolyze ATP. The movement of Rho along the RNA is coupled to ATP hydrolysis, and the interactions among RNA, Rho, and ATP generate a processive directional movement.

Rho shares considerable sequence similarity and identity with the beta -subunit of F1-ATP synthase (17-20), and secondary structure predictions for each protein are similar (21). A structural model of Rho based on the crystal structure of the alpha -, beta -, and gamma -subunits of F1-ATP synthase has been used to describe its function, including the binding of the antibiotic, bicyclomycin (20, 22, 23). F1-ATP synthase has five distinct subunits, alpha , beta , gamma , delta , and epsilon , in which three alpha - and three beta -subunits form a toroid ring (24) similar to Rho (25, 26). The beta -subunits are catalytically active, whereas the alpha -subunits bind ATP much tighter than the beta -subunits and do not support ATP hydrolysis (synthesis) (27). The three beta -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). This potential drives the rotation of the gamma -subunit. As the gamma -subunit turns, it ratchets the switch between the three conformational states of the beta -subunits. The release of ATP is the energy-requiring, rate-limiting step (30). Rho, unlike F1-ATP synthase, is a homohexamer and hydrolyzes ATP to drive its 5' to 3' translocation along the RNA. Unlike F1-ATP synthase, hydrolysis of ATP is converted to translational motion not rotary motion. Movement of Rho is proposed to occur without rotation because rotation would presumably coil the RNA and impede translocation (31).

We have proposed previously (14) a detailed translocation mechanism whereby Rho moves along the RNA by binding RNA at two separate loci in the central hole. Unidirectional translocation requires more than one RNA-binding locus; binding between these sites is ordered and sequential, and the translocation is highly processive. RNA interaction at one locus is proposed to alternate between tight and loose binding phases, whereas RNA at the other site binds but in the opposite phase. Alternating RNA binding at each locus is postulated to be the result of ATP binding, hydrolysis, and release of ADP and phosphate within a subunit pair.

Critical to elucidating the tracking mechanism of Rho is a detailed understanding of ATP binding and hydrolysis as it relates to movement along the RNA. Recently, several groups have reported [gamma -32P]ATP binding to Rho using Scatchard binding analysis. These reports have differed as to the number and types of ATP-binding sites. One argument holds that only three ATP-binding sites are present in Rho (32, 33), and ATP is sequentially hydrolyzed (34). Other data suggest Rho has three tight and three loose ATP-binding sites, wherein the proposed ATP catalytic site exhibits loose binding and the control site exhibits tight binding, similar to the alpha -subunit of F1-ATP synthase (35-37). These conclusions were based, in part, on the similarity of Rho and F1-ATP synthase as a model for ATP binding and hydrolysis in which certain adenosine trinucleotides remained tightly bound during conditions of continuing hydrolysis (37). However, these finding have not been reproduced in other laboratories (33). Still another group suggests that there are two types of ATP-binding sites that provide three strong and three weak loci (38). Measured by affinity constants, 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) Furthermore, these authors suggested that ATP permanently bound to a tight site would block the reaction cycle.

Previous determinations of the Kd for ATP binding were carried out in the absence of RNA where Rho is locked into a non-catalytic, hydrolytically inactive conformation. ATP binding has not been measured in the presence of RNA because RNA activates Rho and prevents thermodynamic equilibrium measurements. ATP hydrolysis is rapid; most of the ATP is hydrolyzed to ADP and phosphate in the time it takes to measure bound ATP (33, 37). Furthermore, the filter binding techniques that were used in some of these studies often did not accurately measure loosely bound ATP. Rinsing or washing the filter removes weaker binding ATP with adventitiously bound ATP. Thus, corrections were made in some cases to account for spurious ATP binding. To measure ATP binding to Rho more accurately, we mutated phenylalanine 355 (Phe-355) to tryptophan (F355W) (14). Based on sequence alignment and the structural model of Rho (31), Phe-355 is analogous to beta -Tyr-331 of the E. coli F1-ATP synthase. The mutation beta -Y331W in F1-ATP synthase allowed ATP binding to be measured by fluorescence quenching (39-41).

Here we document ATP and ADP binding in terms of their dissociation constants derived from fluorescent quenching in the F355W mutant. We also measure the effects of poly(C) and bicyclomycin on the nucleotide dissociation constants. The changes on ATP dissociation constant are discussed in terms of the mechanism of Rho translocation 5' to 3' along RNA.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Enzymes-- Bicyclomycin was kindly provided by Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan), and was further purified by three successive silica gel chromatographies using 20% methanol/chloroform as the eluant (42). Oligonucleotide primers were synthesized by Genosys Biotechnology, Inc. (The Woodlands, TX). T4 polynucleotide kinase, T4 DNA ligase, and restriction enzymes were purchased from Promega Co. (Madison, WI). Pfu DNA polymerase was obtained from Stratagene (La Jolla, CA). The metal chelating column was from Amersham Biosciences.

Radionucleotides [gamma -32P]ATP and [alpha -32P]CTP (3000 Ci mmol-1) were purchased from PerkinElmer Life Sciences, and nucleotides and RNase inhibitor were from Ambion, Inc. (Austin, TX). Polyethylenimine thin layer chromatography plates used for ATPase assays were purchased from J. T. Baker, Inc. (Phillipsburg, NJ). Ribo(C)10 was obtained from Oligos, Etc. (Wilsonville, OR), and ATP analogues were from Sigma. All other chemicals were reagent grade.

Generation of Rho Mutants F355W/W381Y and W381Y-- The plasmids pET-RhoW (wild-type Rho) and pET-355W (F355W Rho) were used as templates for DNA amplification. Overlapping primers with the desired mutation (W381Y) forward, 5'-gaactgcagaaaatgtatatcctgcgcaaaatc-3', and reverse, 5'-gattttgcgcaggatatacattttctgcagttc-3', were used to introduce base changes, as described in the QuikChangeTM site-directed mutagenesis kit (Stratagene). The resulting PCR-amplified plasmid DNA was digested with DpnI and transformed into host strain JM109. Isolated plasmid DNA from the transformed cells was sequenced to identify specific site changes. The entire rho gene was sequenced to ensure no other mutations were present in the mutated genes. The F355W Rho mutant was generated from pET-RhoW (14).

Rho Activities-- Assays for poly(C)-dependent ATPase activity and poly(dC)-ribo(C)10-dependent ATPase activity were carried out at 32 °C, and transcription termination was carried out at 37 °C as described (20).

Isolation of Rho-- Expression of the mutant Rho proteins was carried out by transforming the pET plasmid into the salt-induced T7 polymerase host BL21-SI (Invitrogen). Cells were grown on NaCl-free LB media at 37 °C to an absorbance of 0.5 at 550 nm and induced by the addition of 0.3 M NaCl. Rho was isolated from 500-ml cultures induced for 4-10 h. The induced cells were collected by centrifugation at 10,000 × g and suspended in 20 ml of 50 mM Tris-HCl (pH 7.6), 5% glycerol, 0.23 M NaCl, and 0.1 mM DTT.1 Phenylmethylsulfonyl fluoride was added to 23 µg/ml, and lysozyme was added to 130 µg/ml. After digestion for 1 h at 25 °C, the suspension was briefly sonicated using a Branson probe sonicator and was centrifuged at 17,000 rpm for 30 min. The clear yellow supernatant was placed directly on a metal chelating column (Amersham Biosciences) equilibrated with nickel sulfate as per the manufacturer's instructions. The column was washed with 10 ml of the wash buffer (0.02 M sodium phosphate, 0.5 M NaCl, pH 7.2) and then with another 10 ml of wash buffer containing 10 mM imidazole, and Rho protein was eluted using an imidazole gradient from 10 to 500 mM in wash buffer. Protein concentrations were measured using the BCA protein assay as described (43).

Equilibrium Binding of [gamma -32P]ATP to Rho-- Equilibrium binding of ATP to F355W and His-tagged wild-type Rho was measured at room temperature (22 °C) using the nitrocellulose membrane binding assay as specified (35) with some modifications. The nitrocellulose membrane circles (25 mm) were treated with 0.5 N NaOH, rinsed extensively with distilled water, and equilibrated in the binding buffer (40 mM Tris-HCl (pH 7.9), 12 mM MgCl2, 50 mM KCl, 0.1 mM dithiothreitol) before use. The reactions (50 µl) contained 40 mM Tris-HCl (pH 7.9), 12 mM MgCl2, 50 mM KCl, 0.1 mM EDTA, 0.1 mM dithiothreitol, 400 nM Rho hexamer, 400 µM bicyclomycin, and various concentrations (0.5-300 µM) of ATP mixed with [gamma -32P]ATP were incubated at room temperature (~10 min) and then filtered through the nitrocellulose membrane assembly. The membranes were washed with 1 ml of binding buffer with 400 µM bicyclomycin before and after filtration. The radioactivity on the membrane was quantitated using a scintillation counter (Beckman LS 6000SC).

Fluorescence Quenching by ATP-- The fluorescence of F335W and wild-type Rho was carried out in a Cary Eclipse spectrofluorimeter at 22 °C using 400 nM Rho hexamer in 40 mM Tris-HCl (pH 7.9), 50 mM KCl, 12 mM MgCl2, 0.1 mM EDTA, and 0.1 mM DTT with a stirred cuvette. Emission spectra were recorded between 300 and 450 nm with excitation at 280 nm and zeroing at 300 nm, which allowed the comparison of spectra. Fluorescence quenching in the absence of RNA was measured by the addition of ATP to the stirred cuvette, waiting 1 min, and recording the spectra. ATP or ADP was added in range from 0.1 to 600 µM in the absence of poly(C). The ATP/ADP titrations were also conducted in the presence of 400 µM bicyclomycin. The non-hydrolyzable ATP analogues, gamma -S-ATP, AMP-PNP, and AMP-PCP, were used to titrate the fluorescence quenching of F355W in the absence and presence of 400 µM bicyclomycin. The extent of fluorescence quenching in the presence of poly(C) was measured using the spectrofluorimeter in the kinetic mode. Rho plus poly(C) was premixed and diluted into a fluorescence cuvette with stirring. Excitation was at 280 nm, whereas emission was measured at 350 nm as a function of time. After ATP addition (10 s), a new fluorescence base line was established, and the percent of quenching was measured using 600 µM ATP at 100%.

Stop-flow Determination of the kon for ATP-- The second order rate constants for the binding of ATP to Rho (240 nM hexamer) were measured at 10 °C for each of five ATP concentrations (5, 10, 30, 60, and 120 µM) using an SFA-20 Rapid Kinetics Accessory and Pneumatic Drive (Hi-Tech Scientific, Salisbury, UK) stop-flow attachment with a fluorescence cuvette inserted into the Cary Eclipse spectrofluorimeter. The measurements were repeated in the presence of either 20 nM poly(C) or 80 µM bicyclomycin. Fluorescence quenching at 350 nm was measured in kinetic mode at 10 °C using a circulating water bath to maintain temperature of the stop-flow syringes and the measuring cuvette. Rho, Rho and poly(C), or Rho and bicyclomycin were placed in one syringe and ATP in the other. Fluorescence quenching using 600 µM ATP was assumed to completely quench Rho fluorescence. Initial rates were measured in triplicate for each ATP concentration and reported as average ± S.D. The overall average is the average of all 15 measurements ± S.D.

Determinations of Sedimentation Coefficients-- Sedimentation coefficients were carried out as described (44) with some modifications. Samples containing 4, 40, and 400 nM Rho hexamer in the absence or presence of 1 or 600 µM ATP in 400 µl of sedimentation buffer (40 mM Tris-HCl (pH 7.9), 50 mM KCl, 12 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 5% glycerol) were layered on the top of a 5-ml 10-30% linear glycerol gradient. For samples containing ATP, the same ATP concentrations were present in the glycerol gradients. Sedimentation markers were 400 µl of 80 µg of bovine liver catalase (11.15 S), 400 µl of 30 µg of calf intestinal mucosa alkaline phosphatase (6.23 S), and 400 µl of 225 µg of bovine hemoglobin (4.3 S) and were layered on the glycerol gradient in separate tubes. After centrifugation at 45,000 rpm for 16 h at 4 °C in a Beckman SW50.1 rotor, 200-µl fractions were collected. The fractions were assayed as described (44). For ATPase assays, 10 (for 4 nM Rho hexamer) or 2.5 µl (for 40 and 400 nM Rho hexamer) of each fraction was added to ATPase buffer containing 40 mM Tris-HCl (pH 7.9), 50 mM KCl, 12 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 0.25 mM ATP, 0.1 µCi of [gamma -32P]ATP, and 40 nM poly(C). The reaction was allowed to proceed 20, 10, and 1 min for 4, 40, and 400 nM Rho hexamer, respectively. Phosphate release was measured on the PhosphorImager plates by using Fuji BAS 1000 BioImaging Analyzer. Marker proteins were detected as described (44).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Properties of His-tagged F355W Rho-- The Km and Vmax values for His-tagged F355W Rho were compared with wild-type His-tagged Rho to determine whether the change from phenylalanine to tryptophan adversely altered Rho function. The poly(C)-dependent ATPase activity at saturating ATP concentrations showed that F355W had a kcat of 1000 min-1 compared with 2380 min-1 for wild-type, and F355W had a Km for ATP of 83 µM compared with wild-type of 58 µM. The kcat for poly(dC)-ribo(C)10-dependent ATPase activity with saturating concentrations of ATP and varying ribo(C)10 for F355W was 500 min-1 compared with 850 min-1 for wild-type, and the Km for ribo(C)10 for F355W was 26 µM compared with 9.1 µM for wild-type. Transcription termination efficiencies for F355W were 96% that of wild-type (14). We concluded from these data that the substitution of tryptophan for phenylalanine did not significantly alter the kinetic properties of Rho and that F355W was a reasonable mutant of Rho to study ATP binding.

ATP Titration of Fluorescence Quenching-- Fluorescence quenching of F355W Rho as a function of ATP concentrations measured at room temperature (22 °C) is seen in Fig. 1A. The residue Phe-355 is projected to be base stacked to the adenosine ring of ATP (14), as predicted from the crystal structure of F1-ATP synthase (27). This notion is supported by substitution of the analogous residue in E. coli F1-ATP synthase, beta -Tyr-331 to tryptophan (40, 41, 45). Fig. 1B shows the change in fluorescence quenching of F355W at 350 nm as a function of ATP concentration. The curve could only be fitted to a two-component exponential decay suggesting that two different binding sites were present. A Scatchard plot of the ATP-induced fluorescence quenching of F355W showed a biphasic curve instead of a straight line for the binding of ATP (Fig. 2, ). However, on the basis of the data generated from the Scatchard plot, three assumptions were made, and they require validation. The first is that all the fluorescence quenching arose from the binding of six molecules of ATP per hexamer. The second is that Trp-381 did not contribute to the fluorescence quenching by ATP. The final assumption is that Rho in solution was hexameric and not a combination of monomers and dimers.


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Fig. 1.   Fluorescence quenching of mutant Rho F355W (400 nM hexamer) as a function of ATP concentration. A, emission spectra at varying ATP concentrations measured at 22 °C. ATP concentrations are as follows: 0, ; 0.5, open circle ; 1, black-down-triangle ; 5, down-triangle; 10, black-square; 15, ; 20, black-diamond ; 30, diamond ; 50, black-triangle; 75, triangle ; 100, ; 150, hexagon ; 200, ; 300, open circle ; 400, black-down-triangle ; 500, down-triangle; and 600, black-square µM. The emission spectra were measured at an exciting wavelength of 280 nm, and each spectrum was zeroed at 300 nm before recording. B, a plot of the change of quenching at 350 nm as a function of ATP concentration. The curve could only be fitted using a two component exponential decay (solid line).


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Fig. 2.   Scatchard analysis of ATP binding to F355W Rho. Scatchard analysis of ATP or ADP binding to Rho treated with poly(C) or bicyclomycin. , Rho titrated with ATP; black-square, Rho titrated with ADP; black-triangle, Rho titrated with ATP in the presence of 400 µM bicyclomycin; triangle , Rho titrated with ADP in the presence of 400 µM bicyclomycin; and black-diamond , Rho titrated with ADP in the presence of 100 nM poly(C).

Stoichiometry of ATP Bound to Rho in the Presence Bicyclomycin-- The number of ATPs bound to His-tagged wild-type hexameric Rho in the absence or presence of 400 µM bicyclomycin was determined by nitrocellulose filter binding at 22 °C and correlated to fluorescence changes in F355W. In the absence of bicyclomycin, 3.1 ATP molecules per hexameric Rho was obtained in agreement with values reported (32, 33). In the presence of bicyclomycin, however, an ATP binding stoichiometry of 5.0 ATP molecules per hexameric Rho was obtained (Fig. 3A). This experiment was repeated for wild-type Rho and shows the binding of ATP in the presence and absence of 400 µM bicyclomycin (Fig. 3B). The presence of bicyclomycin caused an increase in the ATP affinity for Rho and diminished the loss of its binding at weaker binding sites that may have occurred by washing. The binding of radiolabeled [gamma -32P]ATP to F355W in the presence of bicyclomycin was used to correlate fluorescence quenching to ATP binding and to corroborate the notion that 600 µM ATP corresponded to complete quenching of fluorescence by six ATPs binding to Rho.


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Fig. 3.   Equilibrium binding of ATP to bicyclomycin-treated F355W Rho and wild-type Rho. A, Rho protein was titrated at 22 °C with [gamma -32P]ATP in the presence of 400 µM bicyclomycin as described under "Experimental Procedures." Binding was determined by nitrocellulose filter binding. B, His-tagged wild-type Rho (400 nM hexamer) was titrated at 22 °C with [gamma -32P]ATP in the presence of 400 µM bicyclomycin () and in the absence of bicyclomycin (open circle ).

The fluorescence quenching by Scatchard analysis indicated two distinguishable ATP-binding sites on Rho hexamer (Fig. 2, ). The first site binds three ATPs with a Kd1 of 3.0 ± 0.3 µM (tight binding site), and the second binds three ATPs with a Kd2 of 58 ± 3 µM (loose binding site). In the presence of 400 µM bicyclomycin, the Kd1 decreased to 1.4 µM, whereas the Kd2 decreased only slightly to 51 µM as summarized in Table I. The number of each type of binding site remained constant, which suggested that bicyclomycin affected both binding sites making them bind ATP even tighter but that the greatest effect was on the tight ATP-binding site.


                              
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Table I
The dissociation constants and stoichiometry of ATP binding to hexameric F355W Rho
The concentrations are as follows: F355W Rho, 400 nM hexamer; bicyclomycin (BCM), 400 µM; poly(dC), 120 nM; poly(C), 100 nM. Measurements were conducted at 22 °C.

ADP-induced fluorescence quenching of F355W showed that the 5.5 ADP-binding sites had a Kd2 of 91 µM and 0.5 sites had a Kd1 of 1.1 µM (Fig. 2, black-square). ADP binding in the presence of bicyclomycin (400 µM) did not alter this pattern; the 5.5 binding sites per Rho hexamer exhibited a Kd2 of 84 µM, and the 0.5 sites had a Kd1 of 1.3 µM. However, ADP binding to Rho in the presence of poly(C) gave a single Kd (all six sites) of 175 µM, showing that ADP binding was much weaker in the presence of poly(C) (Fig. 2, black-diamond ).

Fluorescence Quenching from Trp-381 in His-tagged Wild-type Rho-- The Rho mutant, F355W, contains a second tryptophan, Trp-381, that is present in wild-type Rho. Fluorescence quenching of wild-type Rho was measured to determine the extent of interference Trp-381 fluorescence had on the ATP-induced fluorescence quenching of F355W. The addition of ATP to wild-type Rho (Trp-381) at concentrations below 100 µM resulted in ~6% of the total quenching seen in F355W. These data indicate that the fluorescence quenching of Trp-381 by ATP had a negligible effect on the measured Kd1 of F355W. However, at higher ATP concentrations (100-600 µM) about 30% of the total quenching was observed. A Scatchard plot of the fluorescence quenching from 0 to 600 µM ATP shows biphasic behavior (Fig. 4). It was assumed that at 600 µM ATP six nucleotides are bound to Rho, and this accounts for the quenching. Although a relatively small percentage of quenching was seen below 100 µM ATP, a lower Kd could be calculated for the binding of (presumably one) ATP. Preliminary experiments using a fluorescence-labeled bicyclomycin suggest that a conformational change in Rho upon ATP addition may be responsible for quenching at ATP concentrations below 100 µM (data not shown). Above 100 µM ATP, quenching is thought to occur through nonspecific interactions.


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Fig. 4.   The Scatchard plot of the fluorescence quenching of wild-type Rho (Trp-381).

The possibility that this quenching could interfere with the determination of the Kd2 for the Rho F355W mutant was examined. The contribution of the fluorescence quenching from His-tagged, wild-type Rho was subtracted from the observed quenching of F355W Rho from matching concentrations determined by the BCA protocol and then plotted as a Scatchard plot. Although the fluorescence intensity of F355W was twice that of wild-type in the absence of ATP, the resulting Scatchard plot was not linear (data not shown) indicating more than one Kd for ATP binding. The Kd1 values are not likely to be altered by ATP quenching arising from Trp-381, but the Kd2 values are subject to some uncertainty. The ATP-induced fluorescence quenching arising from Trp-381 may lead to higher Kd2 values. Table I summarizes ATP binding under various conditions determined from the F355W fluorescence quenching without correction for Trp-381 quenching.

The Fluorescence Quenching of the Double Mutant F355W/W381Y by ATP-- To remove background fluorescence quenching that arose from Trp-381, the double mutant F355W/W381Y was generated. The poly(C)-stimulated ATPase hydrolysis gave a kcat of 500 min-1 and a Km for ATP of 125 µM. The kcat is about 20-25% of the wild-type Rho, whereas the Km value increased from 58 to 125 µM. Addition of 100 µM ATP to this double mutant resulted in the complete quenching of the F355W fluorescence (Fig. 5A). Titration of the fluorescence quenching with ATP (0-100 µM) produced biphasic Scatchard plots with three tight and three loose ATP-binding sites (Fig. 5B). However, the Kd1 was ~100-fold lower (tighter) than that measured for the single F355W mutant, and the upper limit of Kd1 could only be estimated from the Scatchard plot (Table II). The Kd2 values also showed tighter binding and ranged from 35- to 200-fold lower than seen for F355W. Because we observed two ATP binding events and the Kd values are much lower than seen for the single mutant, F355W, we concluded that both the Kd1 and Kd2 we observed for the single or double mutant represented two specific ATP binding interactions.


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Fig. 5.   Fluorescence quenching of double mutant Rho F355W/W381Y (400 nM hexamer) as a function of ATP concentration. A, emission spectra at varying ATP concentrations is as follows: 0, ; 0.125, down-triangle; 0.25, black-square; 0.5, diamond ; 1, black-triangle; 2, hexagon ; 3, ; 4, down-triangle; 5, black-square; 7.5, diamond ; 10, black-triangle; 15, hexagon ; 20, ; 30, down-triangle; 50, black-square; 75, diamond ; and 100 black-triangle µM ATP at 22 °C. The emission spectra were measured at an exciting wavelength of 280 nm and zeroed at 300 nm before recording. B, the Scatchard plot of the fluorescence quenching at 350 nm as a function of ATP concentration.


                              
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Table II
The dissociation constants and stoichiometry of ATP binding to hexameric Rho mutant F355W/W381Y
The concentrations were as follows: F355W/W381Y Rho, 400 nM hexamer; bicyclomycin (BCM), 400 µM; poly(dC), 120 nM; poly(C), 100 nM. The measurements were conducted at 22 °C.

The Effect of RNA on ATP Binding to F355W Rho-- RNA induces Rho ATP hydrolysis; in the absence of RNA, Rho is a static, inactive enzyme. Therefore, we asked the question of whether poly(C) binding alters the extent and stoichiometry of ATP binding. These studies were carried out using the non-hydrolyzable ATP analogues, gamma -S-ATP, AMP-PNP, and AMP-PCP, to minimize ATP analogue hydrolysis. Thus, Rho and poly(C) were premixed and placed in the fluorescence cuvette (22 °C), and the ATP analogue was added while fluorescence at 350 nm versus time was measured. Upon ATP analogue addition, quenching was rapidly seen, and within 10 s a new, stable base line was established. We estimated that less than 1% of the ATP was hydrolyzed in this time. In the absence of RNA, gamma -S-ATP binds similarly to ATP (Table III). We observed three tight (Kd1 = 0.2 µM) and three loose (Kd2 = 70 µM) ATP-binding sites per hexamer, and in the presence of bicyclomycin, we found three tight (Kd1 = 0.1 µM) and three loose (Kd2 = 50 µM) gamma -S-ATP-binding sites per hexamer. In the presence of poly(C), gamma -S-ATP binding remained at three tight (Kd1 = 0.9 µM) and three loose (Kd2 = 88 µM) binding sites per hexamer. The increase in Kd1 for ATP binding when poly(C) was added was larger than the increase in Kd2. Two other ATP analogues, AMP-PNP and AMP-PCP, gave varying results; both bound weaker than ATP and behaved like ADP rather than ATP. AMP-PCP in the absence of poly(C) had two tight and four loose binding sites and changed to zero tight and six loose in the presence of poly(C) with a large increase in the Kd2 values (Table III). The addition of poly(dC) (120 nM) to Rho caused little change in the ATP binding except that Kd1 decreased (Table I).


                              
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Table III
The dissociation constants and stoichiometry of non-hydrolyzable binding of ATP analogues to hexameric F355W Rho
The concentrations were as follows: Rho, 400 nM hexamer; bicyclomycin (BCM), 400 µM; poly(C), 100 nM. The measurements were conducted at 22 °C.

Sedimentation Assay-- At 400 nM Rho hexamer, the concentration used in the fluorescence quenching experiments, Rho should exist in the hexameric state (25, 26). However, many factors influence hexameric formation including ionic strength, RNA, and ATP binding (25, 44). Two different binding affinities for ATP may arise from a change in the aggregation state upon ATP binding or binding to a dimer instead of a hexamer. To eliminate this possibility, sedimentation coefficients for Rho were measured by glycerol gradient centrifugation (44). The extent of Rho migration was measured using poly(C)-dependent ATPase activity from separated glycerol gradient fractions, and sedimentation coefficients were calculated by comparison with the migration distances of marker proteins. At a concentration of 400 nM hexamer, Rho had a sedimentation coefficient slightly larger than 11.15 S (the sedimentation coefficient of the standard bovine liver catalase) in the absence and presence of ATP (1 or 600 µM) (Fig. 6), indicating that Rho exists as a hexamer under these conditions (25). At 4 and 40 nm concentrations of Rho, the sedimentation coefficients were 4.3 S and 7.2 S, respectively.


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Fig. 6.   Sedimentation studies of Rho. Sedimentation was measured in glycerol gradients. Rho activity was measured in separated aliquots and compared with standards run on separate gradients in the same experiment. The arrows indicate the position of the standards: bovine hemoglobin (4.3 S), calf intestinal mucosa alkaline phosphatase (6.2 S), and bovine liver catalase (11.15 S).

Rho Shows Negative Cooperativity for ATP Binding-- Using a Hill analysis to review the ATP binding data obtained from F355W fluorescence quenching yielded a plot that indicates ATP binds with negative cooperativity. Low concentrations of ATP show a slope close to 1; however, at concentrations between Kd1 and Kd2 the slope is less than 1 (n = 0.44), and at ATP concentrations near Kd2 the slope returns to 1. The addition of 80 µM bicyclomycin augments this negative cooperativity by causing the intermediate slope to decrease to 0.22 (Fig. 7). The negative cooperativity for ATP binding is a novel finding for Rho, but negative cooperativity is a fairly common occurrence, and the reporting on negative and positive cooperativity is about the same (47).


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Fig. 7.   Hill analysis of the ATP binding to Rho mutant F355W in the presence of 80 µM bicyclomycin.

The kon for ATP-- We measured the kon rate for ATP binding to Rho as a function of ATP concentrations. An average of three measurements employing five ATP concentrations was used to determine the rate, and the experiment was repeated in the presence of 20 nM poly(C) and 80 µM bicyclomycin as shown in Table IV. These experiments were measured at 10 °C. The kon rate for ATP in the absence of poly(C) and bicyclomycin is 3.1 ± 0.40 × 104 M-1 s-1 and increases in the presence of poly(C) to 1.4 ± 0.38 × 105 M-1 s-1. Bicyclomycin in the absence of poly(C) had a minimal effect on these rates marginally increasing the rate to 3.8 ± 0.95 × 104 M-1 s-1. The data were treated as a single exponential decay for each concentration. The kon rate in the presence of poly(C) can be used to estimate the Km for ATP for Rho provided k2, k1, and the dissociation constant, Kd, are known. The ATP hydrolysis rates were measured at 32 °C giving us a Km of 83 µM for ATP and k2 of 17 s-1. The k1 (rate constant for ATP binding) in the presence of poly(C) is 1.4 ± 0.38 × 105 M-1 s-1 at 10 °C. The increase in the rate of Rho ATP hydrolysis with increasing temperature has been measured and is ~2-fold for every 10 °C increase (46). Thus, at 30 °C, the on rate of ATP (k1) would approach 5.6 × 105 M-1 s-1. Because Km = Kd + k2/k1, substituting the values for Kd2 (5.8 × 10-5 M) and k1 and k2 in the equation gives a Km value for ATP of 8.8 × 10-5 M (88 µM). This value is in good agreement with 83 µM determined for F355W and suggests that the weak binding site is responsible for ATP binding at the active site.


                              
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Table IV
Second order rate constants for the binding of ATP to Rho
The concentrations were as follows: Rho, 240 nM monomer; poly(C), 20 nM; bicyclomycin, 80 µM. The measurements were conducted at 10 °C. Data for each ATP concentration is the average of three determinations ± S.D. The final average is the average of all 15 calculations ± S.D.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Six Nucleotide-binding Sites on Hexameric Rho-- The number and type of ATP-binding sites on hexameric Rho have been controversial (33, 37, 38). We have shown that the dissociation constants derived from ATP-induced fluorescence quenching of the Rho mutant F355W (Kd1 = 3.0 µM and Kd2 = 58 µM) in the absence of poly(C) are consistent with three "tight" and three "weak" ATP-binding sites, and the ATP binding data for the F355W/W381Y mutant supports three tight and three loose ATP-binding sites (Table II). These values are close to those reported previously (Ka1 = 3.0 × 106 M-1 or Kd1 = 0.33 × 10-6 M and Ka2 = 0.09 × 106 M-1 or Kd2 = 9.0 × 10-6 M) (38). We show that the dissociation constants for these sites are not influenced by poly(C) (Tables I and II). The dissociation constant for ADP binding to F355W Rho (Kd(ADP) = 92 µM) is similar in all six subunits and is weaker than the Kd2 for ATP. The Kd1(ADP) increases to 175 µM when poly(C) is present (Table I). Bicyclomycin decreased the dissociation constant of the tight ATP-binding site in F355W Rho. This finding is similar to an inhibition mechanism proposed for Rho where inhibition is predicted upon prevention of ATP release at the tight binding site (38). It has yet to be established whether bicyclomycin prevents ATP hydrolysis or the release of either ATP or ADP and phosphate at the tight binding site. The three tight sites we observed are comparable with the three sites reported by Stitt (33). However, Stitt (33) identified only three equivalent high affinity ATP-binding sites (Kd = 0.1-3 µM) that are catalytic. We detect three other weaker binding sites that were missed or dismissed as nonspecific ATP binding. Filter binding experiments using wild-type or F355W Rho, in our hands, showed no plateau in the absence of bicyclomycin, and it was difficult to observe the weaker binding ATP. In the presence of bicyclomycin, the ATP filter binding experiments showed greater than five ATPs bound to hexameric Rho, but these data did not show a definite plateau. Nevertheless, we believe it is reasonable to assume that the stoichiometry is six per hexamer, and for the F355W mutant, we have based our fluorescence quenching on this assumption.

Kim et al. (37) has presented a model with three tight and three weak ATP-binding sites. In this model and in the presence of RNA the three tight ATP-binding sites do not undergo ATP hydrolysis, and the three weak binding sites participate in the fast ATPase turnover. The tight ATP-binding sites are proposed to act like the alpha -subunit of F1-ATP synthase, but unlike Rho, the alpha -subunits lack the specific amino acids required for ATP hydrolysis. The non-hydrolyzable nature of these tight sites was based upon the persistence of bound radiolabeled nucleotides on Rho during ATP hydrolysis (35, 37). Release rate of the radiolabel was 0.02 s-1. However, the ability of Rho to tightly bind adenosine nucleotides under hydrolysis conditions was challenged, and this observation could not be reproduced (33). Analysis of the tight binding [alpha -32P]ATP radiolabel suggests that the bound label was not ATP but more likely ADP (37). We would not expect ADP to bind tightly at all, because the Kd for ADP (92 µM) for F355W Rho is much higher than for ATP, and the Kd for ADP is even higher in the presence of poly(C) (175 µM). The Rho mutant F355W has a Km for ATP of 83 µM, which is incompatible with the Kd1 value (3 µM) for tight ATP binding. By using the Km value of 83 µM ATP, we would predict at 3 µM ATP an ATP hydrolysis rate of about 3.5% Vmax. This analysis implies that ATP binds at the loose site before hydrolysis occurs. Thus, ATP binding at the loose site is a requirement of hydrolysis that occurs on the neighboring subunit, which binds ATP at a tight site. The derived Km from Kd2 and the rate constants k1 and k2 for Rho are in agreement with this notion. Rho may indeed resemble F1-ATP synthase by using "an alternate sites" mechanism by binding ATP at the loose site, is converted to a tight site by ATP hydrolysis on a neighboring subunit, and catalyzes hydrolysis at the newly formed tight site (28). The differences between Rho and F1-ATP synthase is that Rho may require interaction between two subunits where F1-ATP synthase switches between three states: empty, loose, and tight ATP binding.

Wild-type Rho has only one tryptophan (Trp-381); however, fluorescence from Trp-381 is quenched non-specifically by high concentrations by ATP, and this tryptophan is present in the F355W mutant. Whereas the Kd1 is unaffected by fluorescence quenching of Trp-381, there is some ambiguity in the Kd2 value for ATP. The double mutant F355W/W381Y showed two distinct ATP-binding sites, and 100% of the fluorescence was quenched by 100 µM ATP (Fig. 5). The Kd1 was estimated to be less than 0.01 µM, and Kd2 was 1.5 µM (Table II). The same binding trends were observed for the double mutant as seen for the single mutant F355W. ADP binding showed primarily one binding site with a Kd higher than the Kd2 for ATP, which increased in the presence of poly(C). In the presence of bicyclomycin, the Kd1 for ATP decreased, and the Kd2 appeared to decrease only slightly. Initial attempts to measure ATP binding in the presence of poly(C) was done by scanning the fluorescence emission spectrum each time ATP was added. However, ATP was hydrolyzed to ADP and phosphate during the scanning, and we obtained a binding profile similar to that seen with ADP (six loose binding sites with a Kd ~90 µM). By rapidly measuring ATP-induced quenching before significant hydrolysis of ATP occurred, and using a different Rho sample for each ATP concentration, the dissociation constants for ATP binding in the presence of poly(C) was estimated. We measured three tight and three loose ATP-binding sites in the presence of poly(C), and we found these values to be similar to those in the absence of poly(C). The conversion of the tight-to-loose ATP-binding sites by ATP hydrolysis in the presence of poly(C) was inhibited by bicyclomycin. This observation suggested that the six empty ATP-binding sites on Rho are equivalent before ATP was added and does not support the findings of Kim et al. (37) for three persistent (non-hydrolyzable) tight ATP-binding sites during the catalytic cycle. If each ATP site is initially equivalent, then each site is involved in the catalytic cycle of Rho. This finding also differs from Stitt (33) but agrees with the ATP binding data of Geiselmann and von Hippel (38).

Mechanism of RNA Tracking-- We had proposed a model for RNA tracking by Rho based on three non-hydrolytic sites (31). However, this model must now be modified to account for Rho containing six ATP hydrolysis sites that alternate between tight (hydrolytic) and loose ATP binding. Any tracking model must agree with the mechanism of ATP hydrolysis. The mechanism that appears to fit the closest to the data is the alternate sites mechanism first proposed by Geiselmann et al. (38). We expand on this model to include RNA tracking, and we present a minimal dimeric model to assist us; however, Rho is a hexamer, and the mechanism may be more complex. This model assumes sequential hydrolysis of ATP on successive Rho subunits (33, 34). As diagrammed in Fig. 8, ATP hydrolysis depends upon two subunits working cooperatively. The reaction cycle starts after RNA is bound to Rho at the C-terminal tracking site (31). The initial steps of RNA binding and the observation of burst kinetics of ATP hydrolysis (34) will be presented after discussion of the catalytic cycle. Subunit A starts out with RNA binding to the C-terminal domain. At this point ATP is already bound to the tight site labeled T. When RNA binds to the N-terminal subdomain of A (step 1), there is a conformational change to the T* state that activates ATP hydrolysis (step 2). ATP bound to subunit B at the open (Oo) site (step 3) is a prerequisite for the release of phosphate on subunit A (step 4). As phosphate release occurs (step 4), there is a release of energy producing a switch from the T* state to the L* state on subunit A. Concomitant upon the release of phosphate (step 4) from the A subunit, ATP binding on the B subunit goes from open occupied (Oo) (loose) to tight (T) binding (step 5). This switch is felt throughout the Rho hexamer. The hydrolysis also triggers the movement of RNA from the C-terminal subdomain on subunit A to the C-terminal subdomain on subunit B (rate-limiting step). As ADP is released (step 6), RNA is released from the N-terminal RNA binding domain of subunit A (step 7) and binds to the N-terminal RNA binding domain of subunit of B. At this point, the catalytic process repeats. This model then predicts that initial RNA binding to the N-terminal subdomain can lead to the first round of ATP hydrolysis before the RNA can bind to the C-terminal domain leading to a stoichiometric burst of ATP hydrolysis (33, 34). We also predict that ATP is rapidly hydrolyzed at the T site but cannot be released from the tight site until RNA binds to that subunit at both N- and C-terminal domains generating the T* state.


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Fig. 8.   The alternate site model for ATP binding and hydrolysis. The symbols are as follows: T, tight ATP-binding site; T*, tight ATP-binding site activated by RNA binding; L*, loose ADP-binding site in the presence of RNA; Oe, empty or unoccupied site; and Oo, loose occupied ATP binding. The numbers represent the order of events in the mechanism.

Based on our findings, it is proposed that Rho has six ATP-binding sites: three "tight" and three "loose" sites in the absence or presence of poly(C), and these sites synchronously alternate from loose to tight concomitant to the catalytic cycle.

    ACKNOWLEDGEMENTS

We thank Dr. M. Kawamura and the Fujisawa Pharmaceutical Co., Ltd., Japan, for the gift of bicyclomycin and Dr. T. Platt (University of Rochester) for the overproducing strain of Rho.

    FOOTNOTES

* This work was supported by United States Public Health Service Grant GM37934 from the National Institutes of Health (to H. K. and W. R. W.) and the Robert A. Welch Foundation Grant E1381 (to W. R. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biology and Biochemistry, 369 Science and Research Bldg. II, University of Houston, Houston, TX 77204-5001. Tel.: 713-743-8368; Fax: 713-743-8351; E-mail: widger@uh.edu.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212979200

    ABBREVIATIONS

The abbreviations used are: DTT, dithiothreitol; AMP-PCP, adenosine 5'-(beta ,gamma -methylenetriphosphate); AMP-PNP, adenosine 5'-(beta ,gamma -imino)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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