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INTRODUCTION |
The transcription termination factor rho is an essential protein
in Escherichia coli that is required for the release of
certain nascent RNAs from the transcription complex (1-4). The rho
protein contains an ATPase-dependent helicase activity that
can separate the strands of an RNA-DNA duplex. Transcription
termination by rho starts after the rho protein binds to the nascent
RNA transcript at a cytosine-rich entry site (5). The RNA binding event
activates the ATPase activity of rho, which is believed to fuel a
directional 5'
3' movement of the protein along the nascent RNA
chain. Both the ATPase and the helicase activities are necessary
for the rho-dependent termination event (6). Thus, it is
believed that translocation and RNA-DNA duplex unwinding
activities cause disruption of the elongating complex, which results in
transcription termination (7-9).
Several studies of the rho protein have shown that it assembles into a
planar hexagonal structure that contains a central hole (10, 11). The
self-association of rho subunits into a hexamer is reversible in
solution, and stable hexamer is formed in the presence of ATP and/or
RNA (12, 13). Each subunit of rho hexamer contains two functionally
distinct domains, an ATP- and an RNA-binding domain (1). The
ATP-binding domain of rho shows greater than 50% amino acid sequence
homology to the
subunit of F1-ATPase (14). The tertiary
structure of the RNA-binding domain of rho is also very similar to the
N-terminal domain of F1-ATPase, despite little amino acid
sequence homology between these domains of the two proteins (15). A
recent report has also shown similarities among the ATPase mechanism of
rho, the F1-ATPase, and T7 DNA helicase (23, 24). These
observations support the proposal that the structure of the rho hexamer
is likely to be similar to that of the F1-ATPase
(14-16).
Several studies in the literature indicate that the rho hexamer is not
symmetrical. Geiselmann et al. (17) suggested a
D3 symmetry for the rho hexamer, and recently Horiguchi
et al. (18), using extensive chemical cross-linking
experiments, have suggested a C3 or a C3/6
symmetry for the rho quaternary geometry. Equilibrium nucleotide
binding experiments have shown that the rho hexamer binds only three
ATP molecules with a high affinity (19). Others have shown that in
addition to these high affinity sites, there are three low affinity ATP
binding sites on rho (20, 21). Thus, rho protein falls into the class
of hexameric helicases, such as the E. coli DnaB (22) and T7
DNA helicase (23) that were shown to contain two classes of nucleotide
binding sites. The three tight nucleotide binding sites on T7 DNA
helicase hexamer were shown to be noncatalytic, with properties similar
to such sites of the F1-ATPase protein. Because rho protein
is structurally similar to the F1-ATPase protein, we have
investigated the possibility of noncatalytic sites on the rho hexamer.
Our results show that the two classes of nucleotide binding sites on
rho catalyze ATPase turnover at different rates. The ATPase turnover
rate at one class of nucleotide binding sites was about 1500 times
slower than the turnover rate at the other sites. These studies allow a
better understanding of the nature of the two classes of nucleotide
binding sites on the rho hexamer. We propose that the three tight
nucleotide binding sites on the rho hexamer are noncatalytic, serving a
role similar to such sites of the F1-ATPase and T7 DNA
helicase, and that the weak sites are the catalytic sites.
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MATERIALS AND METHODS |
Rho Protein--
Rho protein was overexpressed in E. coli
strain HB101 carrying the rho overexpression plasmid pKS26 (25)
and purified according to Finger and Richardson (26) with slight
modifications. Briefly, protein extracts from heat-induced cells were
subjected to fractionation with Polymin P, ammonium sulfate
precipitation, and successive chromatography on SP-Sepharose FF and
heparin-Sepharose (Amersham Pharmacia Biotech). The final preparation
was stored at
20 °C in rho storage buffer containing 50%
glycerol, 20 mM Tris-HCl (pH 7.9), 100 mM KCl,
0.1 mM EDTA, and 0.1 mM dithiothreitol. Rho
protein concentration was determined by UV absorption at 280 nm using
an extinction coefficient of 0.325 (mg/ml)
1
cm
1 (11).
Nucleotides, RNA Homopolymers, and Other Reagents--
ATP and
RNA homopolymers (poly(C) and poly(U)) were purchased from Amersham
Pharmacia Biotech. Poly(C) RNA had a reported s20,w value of 7.1 in 0.015 M NaCl, 0.0015 M sodium citrate buffer, pH 7.0, with an average length of 420 bases. Poly(U) had an
s20,w value of 6.0, with an average
length of 290 bases. Poly(C) RNA concentration was determined by UV
absorption at 269 nm, using an extinction coefficient of 6200 M
1 cm
1 for the cytosine base,
and poly(U) RNA concentration at 260 nm, with an extinction coefficient
of 9350 M
1 cm
1 for uracil base.
These RNAs were dissolved in TE buffer (40 mM Tris-HCl, pH
7.0, 0.5 mM EDTA) and used without further purification. [
-32P]ATP and [
-32P]ATP were
purchased from Amersham Pharmacia Biotech, and their purity was
assessed by polyethyleneimine
(PEI)1-cellulose TLC and
corrected for in all experiments. The ATP-regenerating reagents,
phosphocreatine and creatine kinase, were purchased from Roche
Molecular Biochemicals.
Equilibrium Binding of ATP to Rho Protein--
Equilibrium
binding of ATP to rho was measured using the nitrocellulose membrane
binding assay. The nitrocellulose membrane circles (25 mm) were treated
with 0.5 N NaOH, rinsed extensively with water, and
equilibrated in the membrane wash buffer (40 mM Tris-HCl,
pH 7.5, 10 mM MgCl2, 50 mM KCl, and
0.1 mM dithiothreitol) before use. The reaction (15 µl)
contained 0.5-500 µM radiolabeled ATP
([
-32P]ATP) in binding buffer (40 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, 50 mM KCl, 0.1 mM dithiothreitol, and 10% (v/v)
glycerol). Rho protein was added to a final concentration of 1.0 µM (hexamer) and the reactions were incubated for 30 s at 18 °C. 12 µl of samples were then filtered through the
nitrocellulose membrane assembly. The membranes were washed with 0.5 ml
of wash buffer before and after filtration. 1-µl aliquots of each
sample were spotted on a separate nitrocellulose membrane to quantitate
total ATP. The radioactivity on the membrane was quantitated on a
PhosphorImager (Molecular Dynamics). The molar amount of ATP bound to
each rho hexamer was determined and plotted versus ATP
concentration. The resulting titration data were fit using a hyperbolic
equation to obtain the apparent Kd, the equilibrium
dissociation constant of rho ATP complex.
ATPase Activity--
The ATPase activity of rho protein was
measured at 18 °C. The rho protein was incubated in the ATPase
reaction buffer (40 mM Tris-HCl, pH 7.8, 100 mM
KCl, 10 mM MgCl2, 0.1 mM
dithiothreitol and 10% (v/v) glycerol) in the presence of the
indicated amount of poly(C) or poly(U) RNA. Trace amounts of a >90%
pure [
-32P]ATP (3000 Ci/mmol), together with
nonradioactive ATP (1.0-3.0 mM), were added to start the
reaction (30-50-µl reaction volumes). At various times, aliquots (5 µl) were withdrawn and quenched with 10 µl of 1 M HCl
and 20 µl of chloroform. Quenched reactions were neutralized by
adding 1 M NaOH, 0.25 M Tris base (about 9 µl), and 1 µl of the quenched reactions was spotted on a
PEI-cellulose thin layer chromatography plate (Whatman). The
radioactive ATP and Pi were separated using 0.3 M potassium phosphate, pH 3.4, as the chromatography
running buffer. The ATP and Pi spots were quantitated using
the PhosphorImager. The initial rates of Pi formation
were determined from plots of molar amounts of Pi versus time of reaction.
Kinetics of Nucleotide Exchange and Identification of the Tightly
Bound Nucleotides--
Either [
-32P]ATP or
[
-32P]ATP was used in this experiment. All the
nucleotide exchange experiments were carried out in an 18 °C room.
Rho protein (0.276 mg/ml, 1.0 µM rho hexamer) was
pre-incubated in the ATPase reaction buffer with a mixture of
nonradioactive ATP (1.0 mM) and radioactive ATP for less
than 10 s, either in the absence or in the presence of RNA. The
reaction mixture was subsequently chased with nonradioactive ATP (10 mM in the same buffer as above). At defined intervals after
addition of the chase ATP, 10-15-µl aliquots were removed and
filtered through pre-wetted nitrocellulose membrane filters. 1 µl of
each sample was spotted on a separate nitrocellulose membrane to
quantify the total ATP. Nonspecific binding of ATP to the
nitrocellulose filters was measured by replacing rho protein with
buffer and corrected in all experiments. The resulting nucleotide
binding/exchange data were fit using nonlinear regression analysis
(SigmaPlot) to obtain the dissociation rate of rho-bound nucleotides.
The chemical form of the nucleotides bound to rho was identified by
extracting the nitrocellulose membrane filters with 0.5 M
perchloric acid, neutralizing with 0.25 M Tris, 1 M NaOH, and analyzing the extracted nucleotides by
PEI-cellulose TLC.
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RESULTS |
Interaction of Rho with ATP--
To investigate the interaction of
rho protein with ATP and to determine the number of ATPs bound to rho
at equilibrium, we have used nitrocellulose filter binding assay. A
constant amount of rho protein was titrated with increasing amounts of
[
-32P]ATP, and the rho-bound ATP was separated from
free ATP by filtration through nitrocellulose membranes. As shown in
Fig. 1, three ATP molecules were bound to
rho at high ATP concentrations. The apparent Kd of
rho-ATP complex was determined to be 17 µM. These results
are consistent with studies that show that although rho has six
identical ATP binding sites, rho binds only three ATPs at equilibrium
(19-21). The ATPs bound to the weak sites are not easily detectable by
this membrane filtration method, possibly because these ATPs dissociate
during the filtration and washing steps.

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Fig. 1.
Equilibrium binding of ATP to rho. Rho
protein (1.0 µM hexamer) was titrated with increasing
[ -32P]ATP (0.5-500 µM) in the absence
of RNA at 18 °C as described under "Materials and Methods." The
binding data were fit to a hyperbola that provided an apparent
Kd of 18.3 ± 0.25 µM and a
maximum of 2.81 ± 0.085 ATPs bound per rho hexamer.
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We then explored the possibility of noncatalytic sites on rho. We
designed experiments to determine whether the ATPs bound at the tight
sites were catalytically competent. The experiment consisted of
measuring the dissociation rate of nucleotides bound to the tight sites
(koff) and comparing this value to the ATPase turnover rate, kcat. If the tight sites are
catalytically competent, then the koff should be
equal to or faster than the steady state kcat.
If the koff from these sites is slower than the
kcat, then the ATPs bound to the tight sites
cannot be catalytically competent and are likely to be noncatalytic.
The ATPase Turnover Rates in the Presence of Poly(C) and Poly(U)
RNAs--
The rho-catalyzed ATPase turnover rate was measured at
18 °C in the presence of poly(C) or poly(U) RNA. The concentrations of RNA and ATP were kept at saturating values; thus the measured velocity would be the Vmax value, which, divided
by the rho hexamer concentration, provided the
kcat. Table I
lists the kcat values obtained in the presence
of poly(C) and poly(U) RNAs. In the presence of poly(C) RNA, the ATPase
kcat was close to 30 mol of ATP hydrolyzed per s
per rho hexamer. Under the same conditions, the poly(U)-stimulated ATPase kcat was only 7.2 mol of ATP hydrolyzed
per s per rho hexamer. This rate appears to be lower because of the
weaker affinity of rho for the poly(U) RNA. Accordingly, the ATPase
kcat increased to 11.4 mol of ATP hydrolyzed per
s per rho hexamer when the poly(U) RNA was increased to 0.33 µg/µl.
The Kinetics of Nucleotide Dissociation from the Rho
Hexamer--
The following experiment was designed to determine the
dissociation rate of the three nucleotides bound to the rho protein at
equilibrium. As shown in Scheme I, the
experiment consisted of preincubating a complex of rho and RNA with
radioactive ATP and then chasing the complex with an excess of
nonradioactive ATP. The radioactive nucleotide bound to rho after
various chase times were quantitated by the nitrocellulose filter
binding assay. The results of such an experiment with poly(C) RNA and
[
-32P]ATP are shown in Fig.
2A. Before chase was added,
there were about 3 nucleotides bound per rho hexamer (Fig.
2A). After chase was added, all of the
-32P-labeled nucleotides dissociated from the rho
protein with a single exponential rate of 0.02 ± 0.002 s
1 (Fig. 2A). A similar nucleotide
binding/exchange experiment, in the presence of poly(U) RNA at 0.2 µg/µl, provided a 10-fold faster nucleotide dissociation rate
constant of 0.15 ± 0.02 s
1 (Fig. 2B).
When the poly(U) concentration was increased about 16-fold, the
dissociation rate decreased to the same level as observed with poly(C)
RNA (0.033 ± 0.005 s
1 at 3.3 µg/µl of poly(U)
RNA). When the experiment was carried out in the absence of RNA (Fig.
2C), 3-4 nucleotides were bound to rho at equilibrium, in
the absence of the chase. After chase was added, the
-32P-labeled nucleotides dissociated from the rho
hexamer at a rate that was too fast to measure manually.

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Fig. 2.
Dissociation kinetics of rho-bound
-32P-labeled nucleotides. The dissociation rate of
radioactive nucleotides bound to rho was measured in the absence and in
the presence of poly(C) and poly(U) RNAs at 18 °C as shown in Scheme
I. A, rho protein (1.0 µM, hexamer) and
poly(C) RNA (0.2 µg/µl) were preincubated and mixed with
[ -32P]ATP (1.0 mM) for about 7 s
before nonradioactive ATP (10 mM) was added as a chase.
After varying chase times (10-400 s), rho-bound radioactive nucleotide
was quantitated by nitrocellulose membrane binding assay. In the
presence of poly(C) RNA, a total of 3.1 ± 0.48 nucleotides (error
calculated from five independent measurements) were bound to rho before
chase was added (shown as at zero chase time). After ATP chase was
added, the rho-bound nucleotides dissociated with an exponential rate
constant of 0.02 ± 0.002 s 1 ( ). B
shows the dissociation kinetics of rho-bound
-32P-labeled nucleotides in the presence of two
different poly(U) RNA concentrations (0.2 µg/µl, shown as , and
3.3 µg/µl, shown as ). About 3-4 nucleotides were bound to the
rho hexamer at both RNA concentrations before ATP chase was added
(shown as , ). The three rho-bound nucleotides dissociated with a
rate constant of 0.15 ± 0.02 s 1 ( ) in the
presence of 0.2 µg/µl RNA and 0.033 ± 0.005 s 1
( ) in the presence of 3.3 µg/µl poly(U) RNA. C, in
the absence of RNA, a total of 4.2 ± 0.54 nucleotides were bound
per rho hexamer before chase was added (shown as ). After ATP chase
was added, the rho-bound nucleotides dissociated at a rate that was too
fast to manually measure ( ).
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Table II compares the
koff values of rho-bound nucleotides and the
rho-catalyzed ATPase turnover rates. In the presence of poly(C) RNA,
the koff of the three tight nucleotides was
about 1500 times slower than the ATPase turnover rate. In the presence of poly(U) RNA, the koff of the nucleotides was
50-340 times slower than the ATPase turnover rate. Thus the
koff of the tightly bound nucleotides was
consistently slower than the RNA-stimulated ATPase turnover rate. These
results indicate that the nucleotides bound at the three tight sites on
the rho hexamer do not participate in the fast ATPase turnover.
Identification of the Slowly Exchanging Nucleotides Bound at the
Tight Sites of Rho--
The above experiments indicated that the ATPs
bound at the tight sites exchanged very slowly with solution ATP. The
next question was whether the tightly bound nucleotides were ATP or
ADP. In other words, does ATP get hydrolyzed at these tight sites? Two experiments were designed to determine the identity (ATP versus ADP) of the slowly exchanging nucleotides. These experiments were carried out in the presence of poly(C) RNA. Essentially the same experiment described above and shown in Fig. 2A was
performed with [
-32P]ATP. After various chase times,
the nitrocellulose membrane that contained the rho-bound radioactive
nucleotide was extracted, and the nucleotides were analyzed by
PEI-cellulose TLC. As shown in Fig. 3,
this experiment showed that most of the nucleotides bound to rho both
before and after addition of the chase were ADP. The total amount of
rho-bound ADP decreased with increasing chase time, as expected. A
control experiment (Fig. 3, lane 9) showed that about one-
third of the radioactive ATP was hydrolyzed with rho and RNA during the
<10-s preincubation period. Therefore, the ATP substrate was not
exhausted during the preincubation period, and the rho-bound
radioactive ADP did not result from rebinding of radioactive ADP from
solution. Similarly, we also determined that the acid extraction
procedure resulted in less than 7% hydrolysis of ATP to ADP (Fig. 3,
lane 11). The above results indicate that the tight sites
are capable of hydrolyzing ATP, but the hydrolysis product dissociates
from the tight sites at a slow rate.

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Fig. 3.
Identification of rho-bound
-32P-labeled nucleotides. The chemical nature of
rho-bound nucleotides in the presence of poly(C) RNA was determined.
The nitrocellulose membranes from the experiment described in Fig.
2A were acid-extracted and the eluted nucleotides were
analyzed by PEI-cellulose TLC (see "Materials and Methods"). The
PhosphorImager scan of the TLC plate is shown. Lane 1 shows
the filter-extracted rho-bound nucleotides before chase was added: ATP,
4% and ADP, 96%. Lanes 2-7 show the filter-extracted
nucleotides after 10, 50, 90, 150, 250, and 400 s of chase
addition. In all the lanes, less than 10% of the total nucleotide was
ATP, and the nucleotide ADP was the major rho-bound species ( 90%).
Lane 8 shows the nucleotides bound nonspecifically to the
nitrocellulose membrane. No nucleotides were detected. Lane 9 shows the composition of nucleotides in the nucleotide
binding/exchange reaction before nonradioactive ATP chase was added.
This control shows that before chase was added the reaction contained
63% ATP and 37% ADP. Lane 10 shows the composition of
nucleotides in a reaction in which ATP was allowed to completely
hydrolyze. Most of the nucleotide is ADP. Lane 11 shows a
control in which radioactive ATP without rho was spotted on the
membrane, which was acid-extracted. This procedure resulted in the
isolation of 89% ATP. Because the commercial radioactive ATP contained
about 4-6% ADP, very little ATP was hydrolyzed by the acid extraction
procedure.
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To confirm these results, a second nucleotide binding/exchange
experiment was carried out with [
-32P]ATP instead of
[
-32P]ATP. If the ATPs are hydrolyzed at the tight
nucleotide binding sites of rho and if the radioactive Pi
does not remain tightly bound, then we should observe no detectable
radioactivity on the nitrocellulose membranes in the nucleotide
binding/chase experiment with [
-32P]ATP. As shown in
Fig. 4, we observed radioactivity bound
to rho which corresponded to about one site per hexamer, before chase was added. This radioactivity was lost after addition of the chase with
the same slow rate constant of 0.02 s
1 as observed in the
[
-32P]ATP experiment. Because the radiolabel on ATP is
in the
position, the slowly exchanging species must be ATP or
Pi. Moreover, because the nucleotide identification
experiment had shown very little rho-bound ATP at the tight sites, we
conclude that about 30% Pi is tightly bound to rho, either
as free Pi or in a complex with ADP, such as
ADP·Pi.

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Fig. 4.
Dissociation kinetics of rho-bound
-32P-labeled nucleotides. The nucleotide
binding/exchange experiment was carried out with
[ -32P]ATP. Rho (1 µM, hexamer) and
poly(C) RNA (0.2 µg/µl) were mixed with [ -32P]ATP
(1.0 mM) for 7 s at 18 °C. Excess ATP (10 mM) was then added, and after varying chase times (10-400
s), aliquots were removed and rho-bound radioactivity was determined by
the nitrocellulose membrane binding assay. A total of 1.37 ± 0.21 sites of rho hexamer had radioactivity bound in the presence of poly(C)
RNA ( ) before chase was added. After ATP chase was added, the
radioactivity bound to rho decreased with a rate constant of 0.026 ± 0.0015 s 1 ( ). In the absence of RNA, 4.28 ± 0.44 sites of rho hexamer had radioactivity bound ( ) before chase
was added. After addition of the chase, the radioactivity decreased at
a rate that was too fast to measure ( ). Asterisk
indicates 32P-labeled nucleotide.
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Cooperativity Between the Catalytic and the Noncatalytic Sites of
Rho--
Results thus far suggested that the ATPase turnover at the
noncatalytic sites was about 1500 times slower than the ATPase turnover
at the catalytic sites. We wanted to investigate whether there was
cooperativity between the catalytic and noncatalytic ATPase sites of
rho. To this end, we measured the ATPase turnover at the noncatalytic
sites by measuring the koff of nucleotides from
the tight sites under two conditions. In one experiment, we measured
the koff under reaction conditions where active
hydrolysis of ATP was not occurring at the catalytic sites. In a second
experiment, we measured the koff under reaction
conditions where hydrolysis was occurring at the catalytic sites. To
satisfy the first condition, a mixture of rho protein and RNA was
incubated with a lower concentration of [
-32P]ATP (0.3 mM). Because of the lower ATP concentration, most of the
ATP was hydrolyzed with RNA in the 2-min period before nonradioactive ATP chase was added, as shown in Fig. 5
(inset). Under these conditions, there were less than three
radioactive nucleotides bound to rho at the tight sites, and these
nucleotides dissociated at a rate that was too fast to manually
measure. To satisfy the second condition, the same nucleotide
binding/exchange experiment was carried out in the presence of an ATP
regeneration system. Fig. 5, inset, shows that in the
presence of the ATP regeneration system, the reaction mixture contained
mostly ATP at the time chase was added. The three radioactive
nucleotides that were bound prior to addition of the chase dissociated
at a slow rate of 0.015 s
1 (Fig. 5). These results
indicate that the three tight sites of rho retain their nucleotides at
the active site as long as there is excess ATP in solution. Rho
undergoes active ATP binding/hydrolysis events at the catalytic sites
when ATP is present in solution. This active ATP binding and hydrolysis
event at the catalytic sites appears to be necessary for retaining the
ADP and Pi at the tight sites. These results suggest
cooperativity among the two types of nucleotide binding sites of rho.

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Fig. 5.
Dissociation kinetics of rho-bound
-32P-labeled nucleotides in the presence and absence of
the ATP regeneration system. Rho (1 µM, hexamer) was
mixed with [ -32P]ATP (0.3 mM) at 18 °C
for 10 s. Subsequently, poly(C) was added and the mixture was
further incubated for 120 s in the presence or in the absence of
the ATP regeneration system containing phosphocreatine (10 mM) and creatine kinase (0.8 mg/ml). Nonradioactive ATP (5 mM) chase was added, and after varying chase times (10-300
s), aliquots were removed and assayed for rho-bound nucleotides. In the
presence of the ATP regeneration system, a total of 2.45 nucleotides
were bound per rho hexamer. After ATP chase was added, these rho-bound
nucleotides dissociated with a rate constant of 0.015 ± 0.003 s 1 ( ). In the absence of the ATP regeneration system,
a total of 1.6 nucleotides per rho hexamer were bound. After ATP chase
was added, all the rho-bound nucleotides dissociated at a fast rate
( ). The inset shows the molar amount of ADP present in
the reaction mixture after various periods of incubation with rho and
RNA in the presence ( ) and in the absence ( ) of the ATP
regeneration system. Asterisk indicates 32P-labeled
nucleotide.
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DISCUSSION |
Rho protein is known to contain two classes of nucleotide binding
sites. About 3-4 ATPs bind to rho protein tightly and 2-3 bind weakly
at equilibrium (20). Very little is known about the significance and
the role of these two classes of ATP binding sites. It has been assumed
that the tight sites are the catalytic sites. The results reported in
this paper clarify the role of the tight ATP binding sites on the rho
hexamer. The findings from this study indicate that the tight
nucleotide binding sites on the rho hexamer are noncatalytic. These
sites are capable of hydrolyzing ATP, but the ATPase turnover at the
noncatalytic sites is slow. The hydrolysis products (ADP and
Pi) remain bound tightly at these sites and dissociate at a
slow rate, relative to the ATPase turnover rate at the catalytic sites.
Thus, the three tight ATP binding sites on rho do not participate in
the fast ATPase turnover.
The possibility of rho protein containing noncatalytic sites was
considered previously by Stitt (19), but no evidence for such sites was
obtained. In these previously reported experiments, rho protein was
mixed with radioactive ATP, and the rho-ATP complex was chased with
nonradioactive ATP and RNA. Three radioactive ATPs were bound to rho
before chase was added, and all of them were hydrolyzed upon addition
of RNA. Based on these results, it was concluded that the three
nucleotides were bound at the catalytic sites. Our results are
consistent with the results of Stitt (19). We have also observed
hydrolysis of ATP at these tight sites. However, when we measured the
dissociation rate of the tightly bound nucleotides, we observed a slow
dissociation of the ADP and Pi species bound to these
sites. Therefore, the three tight sites on rho cannot participate in
the fast ATPase turnover and thus cannot be the catalytic sites.
The exchange of nucleotides at the noncatalytic sites is slow only in
the presence of RNA and when excess ATP is present in solution. In the
absence of RNA or when ATP solution was hydrolyzed to ADP and
Pi, the nucleotide exchange rate was faster. As long as the
catalytic sites are actively binding/hydrolyzing ATP, the noncatalytic
sites show stable binding of ADP and Pi. This suggests cooperation between the noncatalytic and the catalytic sites of rho.
The rate of ATPase turnover at the noncatalytic sites also depends on
the identity and the concentration of the RNA cofactor. The nucleotide
dissociation rate constant was smallest from the rho-poly(C) complex,
intermediate from the rho-poly(U) RNA complex, and fastest from rho
uncomplexed with the RNA. The exact reason for this behavior is not
known. If the nucleotides are bound at the interface of the rho
subunits, as found in the F1-ATPase protein, then one might
be able to provide a rationale for the above behavior, in terms of rho
hexamer stability. A slower rate of nucleotide dissociation may occur
from a more stable rho hexamer. It is known that poly(C) RNA binding to
rho results in a stable rho hexamer (12, 27). In a stable hexamer,
breathing or disruption of the subunit interfaces may be slow or occur
less frequently, and thus nucleotide dissociation will be slower. This
is consistent with the observation that nucleotides dissociate more
slowly from poly(C) RNA-bound hexamer.
If the three tight sites on rho are noncatalytic, then the weak sites
that are not detectable by filter binding or ultrafiltration methods
must be the catalytic sites. By performing ATP binding experiments at
higher concentrations of ATP, Geiselmann et al. (20) were
able to detect 2-3 additional ATP binding sites. Similarly, we have
observed 5-6 ADP·AlF4
species bound
to rho in the presence of RNA (data not shown). Geiselmann et al.
noted that the equilibrium dissociation constant of the three weak
ATP binding sites (Kd ~ 10 µM) was the same as the enzymatic Km of the ATPase activity
(about 10 µM). Furthermore, the three tight sites had a
10-fold tighter Kd value relative to the
Km value (20). These findings support the hypothesis
that the weak ATP binding sites, those sites that hydrolyze and turn
over ATP at a rapid rate, are the catalytic sites. A recent report has
shown that binding and hydrolysis of ATP at the catalytic sites is
sequential, and only one ATP is hydrolyzed at a fast rate (24). This
implies that at any given time there is only one catalytic site that
has a tightly bound nucleotide, and ATP binding at the other two
catalytic sites is weak, because ATP exchange at those sites is rapid.
Noncatalytic nucleotide binding sites appear to be general to the
hexameric helicases, because such sites are now found in at least two
hexameric helicases, rho and bacteriophage T7 DNA helicase. Experiments
similar to the ones reported in this paper were used to show that the
three tight nucleotide binding sites of T7 DNA helicase hexamer are
noncatalytic (23). In T7 DNA helicase, these sites contained dTTP and
some dTDP, and these nucleotides exchanged with a rate constant that
was much slower than the dTTPase turnover rate. Such sites are well
established in the hexameric F1-ATPase protein, which has a
high degree of structural and amino acid sequence homology to rho. The
noncatalytic sites of the F1-ATPase protein bind ATP, and
these ATPs are not hydrolyzed under the conditions of the experiments
(28, 29). In the rho protein, the ATPs at the noncatalytic sites are
hydrolyzed to ADP and Pi, but the hydrolysis products
remain tightly bound and their release into solution occurs very
slowly. Thus, the noncatalytic sites on the hexameric helicases do show
differences from such sites in the F1-ATPase protein. This
is understandable because the structures of these proteins are
different. Rho and T7 DNA helicases are homohexamers and
F1-ATPase is an
3
3
heterohexamer. The
subunits of F1-ATPase are designed
to be noncatalytic. That is, the ATP binding site on the
subunit is
lined with amino acids that facilitate tight ATP binding, but some
critical amino acids that promote efficient hydrolysis of ATP are missing.
In rho, the different rates of ATPase turnovers at the noncatalytic
versus the catalytic sites indicate different conformations of these subunits. These conformational changes do not have to be large
to be significant. Even small changes in the position of the amino
acids at the active sites can be enough to change the microenvironment
of the ATP binding sites and to affect binding and hydrolysis of ATP.
Because rho is a homohexamer, the asymmetry may be induced upon
oligomerization or upon ATP binding/hydrolysis. Based on the similarity
of rho with the F1-ATPase protein, we propose that rho
hexamer may adopt a C3 or a pseudo-C6 symmetry. Such an intrinsic symmetry (C3 or C3/6) has
been proposed by Horiguchi et al. (18) for the rho protein
using chemical cross-linking experiments. In the F1-ATPase
protein, the two classes of sites, catalytic and noncatalytic, are
located on alternating subunits or interfaces between the subunits.
This may be the case with the hexameric helicases as well. The exact
role of the noncatalytic sites in the hexameric helicases or the
F1-ATPase is not clear at present. In helicases, nucleotide
binding at these sites may be necessary for the stabilization of the
quaternary structure of the helicase, or these sites may play a
regulatory function. Alternatively, these subunits may be involved in
nucleic acid binding. There are many questions regarding the role of
these sites that will need further investigation.