 |
INTRODUCTION |
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
-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
-,
-, and
-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,
,
,
,
, and
, in which three
- and three
-subunits
form a toroid ring (24) similar to Rho (25, 26). The
-subunits are
catalytically active, whereas the
-subunits bind ATP much tighter
than the
-subunits and do not support ATP hydrolysis (synthesis)
(27). 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). This potential drives
the rotation of the
-subunit. As the
-subunit turns, it ratchets
the switch between the three conformational states of the
-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 [
-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
-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
-Tyr-331 of the E. coli F1-ATP
synthase. The mutation
-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 |
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 [
-32P]ATP and
[
-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 [
-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 [
-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,
-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 [
-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 |
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,
-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, ; 1, ; 5, ; 10, ; 15, ; 20, ; 30, ; 50, ; 75, ; 100, ; 150, ; 200, ; 300, ; 400, ; 500, ; and 600, 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; , Rho titrated
with ADP; , Rho titrated with ATP in the presence of 400 µM bicyclomycin; , Rho titrated with ADP in the
presence of 400 µM bicyclomycin; and , Rho titrated
with ADP in the presence of 100 nM poly(C).
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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
[
-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 [ -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
[ -32P]ATP in the presence of 400 µM
bicyclomycin ( ) and in the absence of bicyclomycin ( ).
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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.
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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,
). 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,
).
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.
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, ; 0.25, ; 0.5, ;
1, ; 2, ; 3, ; 4, ; 5, ; 7.5, ; 10, ; 15, ; 20, ; 30, ; 50, ; 75, ; and 100 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.
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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,
-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,
-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)
-S-ATP-binding sites per hexamer. In the presence of poly(C),
-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.
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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).
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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).
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.
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DISCUSSION |
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
-subunit of
F1-ATP synthase, but unlike Rho, the
-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 [
-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.
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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.