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INTRODUCTION |
The Escherichia coli Rho protein is a transcription
termination factor that assembles into a hexamer of six identical
protein subunits arranged in a ring (1, 2). The hexamer is the
functional form of the Rho protein, and its biological role is to
release specific nascent RNAs that contain the rut
(Rho utilization) site from the transcription
complex (3-5). The Rho protein is homologous to hexameric helicases,
containing an RNA-dependent ATPase activity and an helicase
activity that can separate the strands of the RNA-DNA duplex in
vitro (6). After the Rho protein initiates at specific sites on
the RNA, it is believed to translocate along the RNA in the 5' to 3'
direction coupled to ATP hydrolysis. The translocation and/or unwinding
activity of Rho results in the disruption of the RNA-DNA duplex in the
elongation complex, which releases the transcript and causes
transcription termination (6-8).
The interactions of the Rho hexamer with the RNA are mediated by two
classes of RNA binding sites (9-12). The primary RNA binding site
residing in the N-terminal domain of Rho polypeptide has affinity for
pyrimidine-rich nucleic acid. The structure of the N-terminal domain of
Rho protein complexed with oligo(rC)9 has been determined
(13). These and other structural studies indicate that the primary RNA
binding sites crown the Rho hexamer and are easily accessible for RNA
binding (2, 13, 14). The RNA has been proposed to wrap around the
primary RNA binding sites of the Rho hexamer (13, 16), consistent with
the protection of 70-80 nucleotides of poly(C) RNA (15). Several
studies indicate that the Rho hexamer contains a secondary RNA binding
site that is distinct from the primary site (9, 11, 12, 17). Based on
mutational and cross-linking studies, and the homology of Rho to the
F1-ATPase, it has been proposed that the secondary RNA binding sites in the C-terminal domain reside within the central channel of hexamer ring (12). This mode of RNA binding is similar to
the mode of DNA binding employed by several hexameric helicases such as
T7 gp4, E. coli DnaB, and T4 gp41 (18-20). In Rho, the interactions of RNA with the secondary sites are necessary for ATPase
activation (9).
The goals of the studies presented here were to determine the kinetic
pathway of RNA binding to the Rho hexamer and to elucidate the roles of
the primary and secondary binding sites in the initiation process. We
have used stopped-flow method to monitor RNA binding in real time by
following the RNA-induced changes in the intrinsic protein fluorescence
of the Rho protein. The observed kinetics indicated a multistep
mechanism for RNA binding. The intrinsic rate constant of each step was
determined by globally fitting the kinetic data at various RNA
concentrations to a four-step model using numerical methods. The
four-step sequential mechanism consisted of a diffusion-limited
bimolecular binding of poly(C) RNA to the Rho hexamer, which we propose
represents interactions with the primary RNA-binding site located on
the outer surface of the ring. The subsequent slower conformational
changes represent ring opening and passage of the RNA strand into the
central channel of the opened Rho ring. Because the ATPase rate is
stimulated only when the RNA interacts with the secondary sites, we
were able to determine the kinetics of RNA binding to the secondary sites by following the presteady-state kinetics of ATP hydrolysis. Conserved mechanisms of binding and initiation were revealed upon comparison of the mechanism of RNA binding of the Rho hexamer with the
DNA binding mechanisms of hexameric helicases such as T7 gp4 (21) and
E. coli DnaB (22).
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EXPERIMENTAL PROCEDURES |
Protein, RNA Homopolymer, Nucleotide, and Buffers--
Purified
Rho protein was a gift from Dr. Katsuya Shigesada (Department of
Genetics and Molecular Biology, Kyoto University, Kyoto, Japan). The
Rho protein was overexpressed in E. coli strain HB101
carrying the Rho overexpression plasmid pKS26 (23) and purified
according to Finger and Richardson (24) with slight modifications. The
Rho protein concentration was determined by UV absorption at 280 nm
using an extinction coefficient of 0.325 (mg/ml)
1 cm
1 (25).
The ATP and RNA homopolymer, poly(C), 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(C) RNA concentration was
determined by UV absorption at 269 nm using an extinction coefficient
of 6,200 M
1
cm
1 for the cytosine base. The RNA was
dissolved in TE buffer (40 mM Tris-HCl, pH 7.0, 0.5 mM EDTA) and used without further purification. [
-32P]ATP was purchased from Amersham Pharmacia
Biotech, and its purity was assessed by polyethyleneimine-cellulose TLC
and corrected for in all experiments. ATP was purchased from Sigma and
used without further purification. Buffer B contains 40 mM
Tris-HCl (pH 7.7), 100 mM KCl, 10 mM
MgCl2, 0.1 mM dithiothreitol, and 10% (v/v) glycerol.
Fluorescence Measurements: Equilibrium RNA Binding--
The
fluorescence spectra (320-400 nm) of Rho protein with and without RNA
or RNA plus ATP were measured at equilibrium with excitation at 290 nm
in a Fluoromax-2 fluorometer (Instruments SA, Inc.) at constant
temperature (18 °C). The fluorescence of Rho protein at 0.1 µM (hexamer) was measured in buffer B. Rho spontaneously
assembles into a hexamer when present at 0.1 mg/ml. In the presence of
RNA or ATP, Rho exists almost exclusively as a hexamer even at 1 µg/ml (26). Hence, the concentration of Rho protein used in this
study (0.1 µM hexamer) is well above the condition
required to form the hexamer in the presence of poly(C) RNA. The
emission spectrum was collected again after poly(C) RNA (0.3 µM) addition and after the addition of RNA and ATP (3 mM). The equilibrium fluorescence measurements were carried
out within 5 min of the addition of RNA. During this time, the amount of ATP hydrolyzed is less than 1.0 mM (30 s
1/hexamer × 300 s × 0.1 µM hexamer). The spectra were corrected for background
fluorescence of buffer B and inner filter effects due to RNA and ATP
absorbencies at 290 nm using the following equation (27),
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(Eq. 1)
|
where Fc,i is the corrected fluorescence
intensity at a particular wavelength, i;
Fo,i is the observed fluorescence intensity;
Ax is the absorbance at 290 nm; and
Am,i is the absorbance at the emission wavelengths.
Stopped-flow Kinetics of RNA Binding--
The stopped-flow
kinetic experiments were performed using an instrument manufactured by
KinTek Corp. (State College, PA). A 40-µl solution containing Rho
(0.2 µM hexamer) with or without ATP (3.0 mM)
in buffer B was rapidly mixed with a solution (40 µl) containing
poly(C) RNA (100-600 nM) at 18 °C in buffer B. The
changes in the intrinsic fluorescence of Rho after mixing with the
poly(C) RNA were monitored by exciting the sample at 290 nm and
monitoring emission above 345 nm using a long pass filter (lp 345 filter from Oriel Corp.). Four to seven kinetic traces were averaged
and fit to the sum of two exponentials (Equation 2) and to the
appropriate RNA binding mechanisms (see below),
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(Eq. 2)
|
where F(t) is the fluorescence intensity
at time t, C is a constant representing the
fluorescence intensity at t =
, A1 and A2 are the fluorescence amplitudes, and
k1 and k2 are the observed rate constants. The 500 data points were distributed in two
time windows to accurately resolve both the fast and slow phases. The
observed rate constants of the fast and the slow phase were plotted
against the poly(C) RNA concentration and fitted to either the linear
equation or the hyperbolic equation (Equation 3),
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(Eq. 3)
|
where kobs is the observed rate at each
[RNA], Vmax is the maximum observed rate
constant, and K1/2 is the concentration of RNA at
which the observed rate is half of the maximum.
Presteady-state ATP Hydrolysis: Three-syringe Rapid Chemical
Quenched-flow Experiments--
The presteady-state kinetics of ATP
hydrolysis were measured in a rapid chemical quenched-flow instrument
built by KinTek Corp. In the three-syringe experiment mode, two delay
times were used (KinTek RQF-3 software). An 8-µl solution of Rho
protein (6.0 µM hexamer) in buffer B was rapidly mixed
with an 8-µl mixture of ATP (2.0 mM) and
[
-32P]ATP (0.10 µCi/ml) for 5 or 10 s at
18 °C. After incubation for 5 or 10 s, the sample was mixed
with 32 µl of poly(C) RNA (2.0 µM) in buffer B added
from the third syringe of the quenched-flow instrument. The
48-µl reactions were incubated in the reaction loop for
varying times (15 ms to 1.5 s) and quenched with 120 µl of 2.0 M formic acid to stop the reaction. Aliquots (1.0 µl) from each acid-quenched reaction at varying time points were spotted on
polyethyleneimine-cellulose TLC, which was developed in 0.4 M potassium phosphate, pH 3.4. The resolved radioactive ATP
and ADP were quantified in a PhosphorImager instrument (Molecular Dynamics, Inc., Sunnyvale, CA). Product formation was equal to the
radioactivity corresponding to ADP divided by the total radioactivity. To estimate ATP hydrolysis in the first delay time, the acid quench was
added from the third syringe instead of the RNA. This control experiment was repeated at least three times, and the values were averaged and taken as the background or hydrolysis at the zero time
point. The efficiency of 2.0 M formic acid as a quenching reagent was also determined by including poly(C) RNA in the collecting microcentrifuge tube under the same setup as performed in the zero time
point control. Less than 0.08% of ATP was hydrolyzed to ADP in both
control experiments, which was the same as the level observed in the
absence of enzyme.
Quantitative Analysis and Global Fitting of the Presteady-state
Kinetic Data--
The intrinsic rate constants were determined by
global fitting of the stopped-flow kinetic data collected at increasing
concentrations of poly(C) RNA. The global nonlinear least-squares
fitting was performed using the software "Scientist" (MicroMath
Research, SLC, UT). After choosing a model for RNA binding,
differential equations were written for each kinetic species in the
mechanism. For global fitting, a separate set of differential
equations, distinguished by different suffixes, were written for each
RNA concentrations. The stopped-flow fluorescence traces were directly fitted by assigning the observed fluorescence at any given time, F(t) as the sum of the background fluorescence
(Fb), which is the free protein fluorescence, and the
fluorescence of each Rho-RNA species (PRi) in the mechanism.
|
(Eq. 4)
|
where Fi is the specific fluorescence of each Rho-RNA
species, and Fb is the background fluorescence due to buffer
and free protein. PRi is the amount of each Rho-RNA species
at any given time, which changes during the time course of RNA binding.
The initial estimates for the rate constants during global fitting of
the kinetic data were obtained from the quantitative analysis of the
observed rate constants versus RNA concentrations. The process of global fitting involved first, fixing the intrinsic rate
constants and determining the specific fluorescence values (Fi). Subsequently, one or the other set of parameters was
kept constant, and global fitting was used to optimize the floating set. Eventually, all the parameters were floated to fit all the data
sets to a single mechanism. The fitting process was governed by a
modified Marquard-Levenberg algorithm making use of the analytical Jacobian matrix. The quality of the fit was judged by visual inspection of overlays of the fitted curves and the data as well as inspection of
the residuals.
The presteady-state ATPase kinetic data were fit to the RNA binding
mechanism in the presence of ATP. Each of the Rho-RNA species
(PRi) was assigned an ATP hydrolysis rate. To
fit the burst of three ATPs/hexamer, an exponential term was included where PR1 species was hydrolyzing ATP. The data fit best when PR1
hydrolyzed three ATPs at the rate of ks = 163 s
1; PR4 hydrolyzed ATP at the RNA-stimulated
rate, kcat = 30 s
1;
and the rest of the species (PR2 and PR3) were hydrolyzing ATP at the
unstimulated rate, ki = 3.0 × 10
4 s
1. Thus, the
formation of ADP at any given time was described by H.
|
(Eq. 5)
|
The presteady-state ATP hydrolysis data provided an additional
constraint to be satisfied by the RNA binding mechanism in the presence
of ATP. Therefore, the resulting intrinsic rate constants in the RNA
binding mechanism in the presence of ATP (see Table II) were obtained
by fitting globally stopped-flow as well as the acid quenched-flow ATP
hydrolysis data.
 |
RESULTS |
We have investigated the transient state kinetics of RNA binding
to E. coli Rho hexamer with the goal of elucidating both the
mechanism of initiation and the roles of the primary and secondary binding sites in RNA binding. We used poly(C) RNA as the ligand because
it is an established substrate that binds to the Rho hexamer with a
high affinity (Kd of 1.0 nM) and
stimulates the Rho ATPase nearly 105-fold (15, 28). To
follow the kinetic pathway of RNA binding in real time, we monitored
the RNA-induced changes in the intrinsic fluorescence of Rho protein
using the stopped-flow method. To determine when the Rho-RNA species
becomes competent in ATPase activity, we measured the presteady-state
kinetics of ATP hydrolysis using the chemical quenched-flow method. The
kinetic data from both types of experiments were globally fit to a
multistep RNA binding mechanism to obtain the intrinsic rate constants
by solving the differential equations using numerical approaches.
Rho Protein Fluorescence Changes Due to Poly(C) RNA
Binding--
The Rho protein contains a single tryptophan and seven
tyrosine residues, and when excited at 290 nm, the Rho protein emits with a broad peak at about 355 nm, as shown in Fig.
1. When poly(C) RNA was added to the Rho
protein without ATP, the fluorescence intensity increased without any
change in its maximum at 355 nm. When poly(C) RNA was added to the
Rho-ATP complex, the fluorescence intensity decreased, without any
change in the maximum. These results indicate that the structures of
the Rho-RNA complexes with and without ATP are different. The observed
fluorescence changes can be used to monitor the real time binding of
poly(C) RNA to Rho hexamer, both with and without ATP.

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Fig. 1.
Fluorescence emission spectra of the Rho
protein. Rho protein (0.1 µM) in buffer B was
excited at 290 nm, and fluorescence emission spectra were measured from
320 to 420 nm at 18 °C. The emission spectrum of Rho alone is shown
by the solid line, Rho protein and poly(C) RNA
(0.3 µM) by the dashed line, and
Rho protein plus poly(C) RNA and ATP (3.0 mM) by the
dotted line.
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Stopped-flow Kinetics of Poly(C) Binding to Rho Hexamer in the
Absence of ATP--
The kinetics of poly(C) RNA binding to the Rho
hexamer were monitored by mixing a solution of Rho (0.2 µM hexamer) with poly(C) RNA (0.3 µM
polymer) in a stopped-flow instrument and by measuring the
time-dependent changes in the intrinsic fluorescence of
Rho. Under the conditions of the experiment, the Rho protein is a
stable hexamer (25). Fig. 2A
shows the resulting trace, where the fluorescence of Rho increases in a
time-dependent manner after mixing with the RNA. The
kinetic trace fits best to the sum of two exponentials rather than one,
as shown by the residuals in Fig. 2A. The fast increase in
Rho protein fluorescence occurred with an exponential rate constant of
148 s
1 and the slower change occurred with an
exponential rate constant of 4.62 s
1. Control
experiments showed no time-dependent protein fluorescence changes when the Rho protein was mixed with the buffer solution.

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Fig. 2.
Stopped-flow kinetics of Rho protein
interaction with poly(C) RNA in the absence of ATP. A,
Rho protein (0.10 µM hexamer) was mixed with poly(C) RNA
(0.15 µM polymer) in buffer B at 18 °C in a
stopped-flow instrument. The time-dependent changes in Rho
protein fluorescence upon excitation at 290 nm were measured. The
kinetics fit best to the two exponential equation (Equation 2) with
rates of the two phases, k1 = 147.6 ± 9.3 s 1 and k2 = 4.62 ± 0.91 s 1 (A1 = 0.242 and A2 = 0.0595) as shown by the residual plots.
B, the kinetic traces of RNA binding were measured at
constant Rho (0.1 µM hexamer) and increasing poly(C) RNA
concentration. The resulting kinetic traces are shown in log time
scale. The solid lines are the fits to the
three-step RNA binding mechanism, shown in Table I. C, the
kinetics traces in B were fit to the sum of two exponentials
(Equation 2). The fast rate constant (k1) was
plotted against poly(C) RNA polymer concentration. The linear increase
occurred with a slope of 7.46 ± 0.72 × 108
M 1 s 1
and an intercept of 11.0 s 1. D,
the slower rate constant (k2) increased
hyperbolically with RNA concentration and fit to a hyperbola (Equation 3) with a K1/2 of 50 nM, a maximum rate
of 4.7 ± 0.6 s 1, and a y
intercept of 1.2 s 1.
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To determine the kinetic pathway of poly(C) RNA binding to the Rho
hexamer, the above stopped-flow experiments were performed at various
RNA concentrations. A constant amount of Rho (0.2 µM hexamer) was mixed with poly(C) RNA at varying concentrations (100-600
nM polymer). The resulting time-dependent
protein fluorescence changes were recorded and shown in Fig.
2B. The kinetic data were analyzed in two ways. First, the
kinetics traces were fit to the sum of two exponentials (Equation 2),
and the exponential rate constants were plotted versus the
poly(C) RNA polymer concentration to obtain the dependences, shown in
Fig. 2, C and D. The analysis of these
dependences provided evidence for a two-step RNA binding mechanism and
provided approximate rate constants for the steps in the pathway.
Second, rigorous analysis was carried out by globally fitting the
kinetic data at varying RNA concentrations to the multistep kinetic
model using numerical approaches, which is described below. The plot of
the observed rate constant of the first exponential phase
(k1) versus [RNA] showed a linear
dependence (Fig. 2C). This dependence indicated that a
stable Rho-RNA species whose fluorescence was greater than free Rho
protein was formed after the bimolecular event. The rate constant for
the bimolecular binding event was estimated from the slope that was
equal to 7.5 × 108
M
1 s
1.
The rate constant of the second phase (k2)
increased in a hyperbolic manner with [RNA] and reached a plateau at
high poly(C) RNA (Fig. 2D). The hyperbolic dependence
provided evidence for a conformational change in the Rho-RNA complex
following the bimolecular encounter. The saturation at about 4.7 s
1 provided an estimate for the rate constant
of this conformational change. Thus, the fluorescence stopped-flow
studies indicated a minimal two-step mechanism for RNA binding
consisting of a bimolecular association between Rho and poly(C) RNA
followed by an isomerization step.
We next attempted to globally fit the kinetic data at various RNA
concentrations to a two-step sequential mechanism using the Scientist
program that uses numerical methods to solve the differential equations
that describe the mechanism. The global fitting procedure is described
briefly under "Experimental Procedures" and in more detail
elsewhere (29). We were not able to obtain a global fit with the
two-step mechanism. Therefore, a three-step mechanism was used as the
model, which provided a good fit with the intrinsic rate constants
shown in Table I. This mechanism showed
that the Rho hexamer interacts with the RNA polymer to form PR1 at a
diffusion-limited rate constant k1 = 7.5 × 108 M
1
s
1, and the complex dissociates with a rate
constant of 12.0 s
1. The collision complex
PR1 isomerizes to PR2 at a forward rate of k2 = 26.2 s
1 and a reverse rate of
k
2 = 2.8 s
1. The PR2
subsequently converts to PR3 at a relatively slow rate, k3 = 5.0 s
1. There was
no detectable rate (k
3) for the conversion of
PR3 to PR2.
Stopped-flow Kinetics of Poly(C) Binding to Rho Hexamer in the
Presence of ATP--
Previous studies have shown that ATP increases
the affinity of Rho protein for RNA (30). Under equilibrium conditions,
in the absence of RNA, the Rho hexamer binds three or four ATP
molecules with very little hydrolysis (31). The kinetics of RNA binding to Rho-ATP complex were therefore measured by mixing poly(C) RNA (0.1 µM) with Rho protein (0.1 µM hexamer, final
concentration) containing 3.0 mM ATP in buffer B, in a
stopped-flow instrument. The fluorescence changes in the Rho protein
upon RNA binding were monitored in the rapid time scale and shown in
Fig. 3A. The initial interactions of the Rho hexamer with RNA led to an increase in fluorescence at a rapid rate, and a subsequent conformational change
decreased Rho protein fluorescence. The increase and decrease in
protein fluorescence with time were fit to the sum of two exponentials with rate constants, 118 s
1 and 17.4 s
1, respectively (Fig. 3A). Note
that the observed rate constants of the fast phases are almost the
same, with or without ATP, but the rate constant of the second phase
with ATP is about 4 times faster.

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Fig. 3.
Stopped-flow kinetics of Rho protein
interaction with poly(C) RNA in the presence of ATP. A,
the Rho protein (0.10 µM hexamer, final concentration)
and ATP (3 mM) in buffer B were mixed with poly(C) RNA
(0.10 µM) at 18 °C in a stopped-flow instrument, and
the fluorescence of Rho was measured after mixing upon excitation at
290 nm. The resulting kinetics showed two phases, and the residuals
show best fit to the sum of two exponentials (Equation 2) with
k1 = 117.6 ± 8.8 s 1 and k2 = 17.4 ± 1.7 s 1 (A1 = 0.31 and A2 = 0.137). B, the RNA binding
kinetics were measured at constant Rho protein (0.1 µM
hexamer) and increasing poly(C) RNA, and the resulting kinetic traces
are shown in the log time scale. The solid black
lines are the fit to the three-step RNA binding mechanism
with ATP, shown in Table I. C, the kinetics traces in
B were fit to the sum of two exponentials (Equation 2). The
fast rate constant (k1) increased linearly with
increasing [RNA] with a slope of 8.6 ± 0.42 × 108 M 1
s 1 and an intercept of 24.4 ± 6.5 s 1. D, the observed rates of the
second phase increased hyperbolically with the RNA concentration and
fit to a hyperbola (Equation 3) with a K1/2 of 57 nM and a maximum rate of 31.5 ± 5.6 s 1.
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To determine the mechanism of RNA binding in the presence of ATP, the
fluorescence stopped-flow studies were carried out at constant Rho and
varying poly(C) RNA concentrations. The final concentrations of the Rho
hexamer and poly(C) RNA were 0.1 µM and 50-200
nM, respectively. The kinetic data were collected up to
1.0 s, and during this period the amount of ATP hydrolyzed was
less than 10 µM. Hence, the concentration of ATP
substrate did not change during the course of our measurements. The
kinetic data (Fig. 3B) were fit to the sum of two
exponentials and globally fit to a kinetic model using numerical
approaches. The rate of the fast phase increased linearly with
increasing [RNA] with a slope of 8.5 × 108
M
1 s
1
(Fig. 3C), which represents the bimolecular rate of RNA
binding to the Rho-ATP complex. Note that this rate is very close to
the rate of RNA binding in the absence of ATP. The rate of the second phase increased in a hyperbolic manner with RNA concentration (Fig.
3D). The hyperbolic fit (Equation 3) provided a maximum rate
of 31.5 s
1, which is about 6 times faster
than the rate measured in the absence of ATP (Fig. 2D).
Thus, ATP has no effect on the initial encounter of the RNA with the
Rho hexamer, but ATP does facilitate the subsequent conformational change.
The RNA binding kinetics with ATP at varying [RNA] also fit best to a
sequential three-step RNA binding model rather than a two-step model.
The global fit was carried out as described above using the program
Scientist. The solid black lines in
Fig. 3B are the resulting fits to the three-step RNA binding
mechanism, shown in Table I. The derived intrinsic rate constants
indicate that the initial bimolecular association of Rho-ATP complex
and RNA result in PR1 with k1 = 8.5 × 108 M
1
s
1 and k
1 = 22.9 s
1. The conversion of PR1 to PR2 occurs at a
rate constant, k2, of 20.5 s
1 and a reverse rate constant,
k
2, of 1.6 s
1, which
is close to the k2 and
k
2 in the absence of ATP. The PR2 is converted
to PR3 at a rate constant, k3, of 31.2 s
1, which is 6 times faster than the rate of
the corresponding step in the absence of ATP. Therefore, ATP does not
affect the first and second steps of RNA binding, but ATP does
accelerate the third step.
The Presteady-state Kinetics of ATP Hydrolysis--
The above
stopped-flow fluorescence studies show that at least three Rho-RNA
species, PR1, PR2, and PR3, are formed during the process of RNA
binding to the Rho hexamer, and the global analysis provides the rate
constants of each step. To fully understand the mechanism of RNA
binding, one however needs to know the structures of the Rho-RNA
complexes that accumulate during the reaction. Unfortunately, at
present, the protein fluorescence changes cannot be interpreted in
terms of the structures of the complexes, and other studies are
necessary. Several studies have shown that Rho contains two classes of
nucleic acid binding sites, the primary and the secondary RNA-binding
sites (11, 32). The primary RNA binding site is accessible, whereas the
secondary site is proposed to lie within the central channel (12). It
is also known that the steady-state ATPase activity of Rho is
stimulated only when RNA interacts with the secondary RNA-binding sites
(9). Thus, RNA needs to travel and bind within the central channel of
the Rho hexamer to activate the steady-state ATPase activity (12, 16).
We therefore measured the presteady-state ATPase activity of the Rho
protein during the period of RNA binding to determine when Rho protein
made stable interactions with the secondary RNA binding sites. These
kinetic studies allowed us to determine the kinetics of RNA binding to
the secondary sites and to speculate on the structures of the
intermediate Rho-RNA species.
The Rho protein was preincubated with a mixture of ATP and
[
-32P]ATP for 5 or 10 s and then rapidly mixed
with poly(C) RNA (Fig. 4A).
The formation of ADP after varying times of incubation with the RNA was
measured, and the resulting data are shown in Fig. 4B.
Initially, three ATPs per hexamer were hydrolyzed at a rate, >150
s
1. This burst of ATP hydrolysis was followed
by a kinetic lag of 0.3 s, during which time no ATP hydrolysis was
observed. The same 0.3-s lag was observed also when the experiments
were carried out at various ATP concentrations ranging from 50 µM to 1 mM. After the 0.3-s lag, ATP was
hydrolyzed with linear kinetics at the expected poly(C) RNA-stimulated
steady-state rate. Comparison of the kinetics of Rho-RNA species
(PRi) formation (dictated by the rate constants determined from
the stopped-flow studies) with the kinetics of ATP hydrolysis, in the
same time period, indicated that the burst of ATP hydrolysis coincided
with the formation of PR1. This suggests that the initial encounter of
poly(C) RNA with the Rho-ATP complex results in the hydrolysis of three
ATP molecules. The subsequent lag in ADP formation indicates that the
PR1 species is not competent in turning over ATP or catalyzing multiple
ATPase turnovers at the RNA-stimulated steady-state rate. The kinetic
lag indicates that the formation of the Rho-RNA species that is
competent in catalyzing multiple turnovers of ATP hydrolysis occurs at
a slower rate.

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Fig. 4.
The presteady-state kinetics of ATP
hydrolysis. A, the diagram shows the design of the
three-syringe acid-quenched experiment. The Rho protein (3.0 µM hexamer) was mixed with [ -32P]ATP and
Mg-ATP (1.0 mM) for 5 or 10 s
(t1) in buffer B at 18 °C. Subsequently, the
Rho-ATP complex was mixed with the poly(C) RNA (1.34 µM
polymer), and the reaction was quenched after varying times
(t2). B, the presteady-state ATP
hydrolysis kinetic experiments were repeated five times, and the
average data are shown. The solid line is the fit
to the four-step RNA binding mechanism, shown in Table II. In this
mechanism, three ATPs per hexamer were initially hydrolyzed by PR1 at a
fast rate of 163 s 1, and only PR4 was capable
of hydrolyzing ATP at the RNA-stimulated rate of 30 s 1. The dashed line
shows the predicted curve for a mechanism in which PR3 and PR4
hydrolyzed ATP at the poly(C) RNA-stimulated rate of 30 s 1.
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The next task was to determine which of the Rho-RNA species, PR2 and/or
PR3, were ATPase-competent. We used the three-step sequential mechanism
of RNA binding and the rate constants derived from the stopped-flow
studies to simulate the ATPase kinetics, as described under
"Experimental Procedures." In the model where PR1, PR2, and PR3
were all capable of hydrolyzing ATP, no lag kinetics were predicted.
When PR2 and PR3 or when PR3 alone was ATPase-competent, then the
predicted lag was too short, as shown by the dashed
line in Fig. 4B. These kinetic simulations
indicated that PR3 was not competent in hydrolyzing ATP and needed to
be converted to PR4 to hydrolyze ATP at the RNA-stimulated rate. Thus,
a PR3 to PR4 isomerization step was added to the three-step mechanism,
and the ATPase kinetics were fit to a four-step sequential RNA-binding
mechanism to obtain the rate of PR3 to PR4 conversion (Table
II). To fit the ATPase kinetics, we
invoked that the first Rho-RNA species, PR1, hydrolyzed three ATPs at a
fast rate, but only the last species in the pathway (PR4) was capable
of catalyzing ATPase turnover at the poly(C) RNA-stimulated rate.
Eventually, both the ATPase kinetics and the fluorescence stopped-flow
kinetic traces, shown in Fig. 3, were fit globally to the four-step RNA binding model to obtain a consistent set of intrinsic rate constants, shown in Table II. The intrinsic rate constants of the first three steps did not change significantly, and the global fit provided the PR3
to PR4 conversion rate of 4.1 s
1.
Kinetic Simulation of the Four-step RNA Binding
Mechanism--
The formation and decay of the various Rho-RNA
intermediates were simulated using the RNA binding mechanism, shown in
Table II, that was derived from the stopped-flow and the ATPase
kinetics. This exercise helps one visualize the formation and decay of
the various Rho-RNA intermediates and to determine which species would accumulate during the reaction. The kinetic simulation (Fig.
5A) shows that PR2 is a
transient intermediate that accumulates to a lesser amount relative to
PR1 and PR3. Based on the finding that the primary sites on Rho are
accessible (2, 32), we propose that PR1 and PR2 are species where the
RNA is bound to the primary binding sites (Fig.
6). The intermediate PR3 accumulates between 0.1 and 0.2 s, and thus the formation of PR4 is delayed by
about 0.3 s. The onset of steady-state ATP hydrolysis activity at
the poly(C) RNA-stimulated rate coincides with the time course of the
appearance of the PR4. Because only PR4 is capable of hydrolyzing ATP
at the poly(C) RNA-stimulated rate, we propose that the RNA in this
species is bound stably to the secondary RNA-binding site in the
central channel of Rho hexamer. Thus, the conversion from PR3 to PR4
involves the passage of poly(C) RNA into the central channel of Rho
hexamer. The process of global fitting also provided information about
the relative fluorescence values of the various Rho-RNA species (Fig.
5B). The kinetic fits predict that PR1 has the highest
fluorescence of all species. The fluorescence of both PR2 and PR3 is
lower than PR1, accounting for the observed decrease in fluorescence,
in the presence of ATP. The fluorescence of the last species PR4 is not
very different from PR3; hence, the last step was not observed in the
stopped-flow experiments.

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Fig. 5.
Kinetic simulation of poly(C) RNA binding to
the Rho hexamer in the presence of ATP. A, the
formation and decay of the various species in the RNA binding pathway
were simulated using the four-step RNA binding mechanism in Table II.
The concentrations of the Rho hexamer and poly(C) RNA were 100 and 150 nM, respectively. The open circles
show the decay of free protein (P) as it binds to the RNA.
The triangles, squares, and diamonds
show the formation and decay of the intermediates PR1, PR2, and PR3,
respectively. The filled circles show the
formation of the final, stably bound Rho-RNA complex (PR4), which is
competent for ATP hydrolysis at a rate of kcat.
B, relative protein fluorescence intensities of each
kinetically distinguishable species are shown by bar
graphs. The total fluorescence of PR1 was set to
100%.
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Fig. 6.
Kinetic mechanism of the hexameric Rho
protein binding to RNA. The model shows the possible intermediate
structures of the Rho protein-RNA complex in the pathway of RNA
binding. The N-terminal RNA-binding domains form a crown on the Rho
hexamer as shown by the smaller lobes. The RNA (R) binds to
this primary site, which is on the outside of ring to form PR1 at a
rapid rate. The RNA fills the continuous clefts exposed on the outside
of the ring to form PR2. The ring opening then leads to passage of the
RNA through the central channel of Rho to form PR3. The ring closes to
form PR4, which renders it competent in ATPase and translocation
activities.
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DISCUSSION |
The Rho protein self-assembles into a hexamer, with or
without ATP, and catalyzes sequence-specific transcription termination in vivo. This process requires initiation or binding of the
Rho hexamer to a specific site on the RNA, followed by unidirectional translocation along the RNA and disruption of the RNA/DNA hybrid in the
elongating complex. Very little is known about the kinetics of
initiation and the mechanism by which RNA binds to the Rho hexamer.
There is ample evidence that both the primary and secondary RNA binding
sites mediate the interactions of the Rho hexamer with the RNA, but the
dynamics of the interactions have not been elucidated. The Rho protein
is homologous to the hexameric helicases, and thus comparative studies
of Rho with other hexameric helicases will also provide general
mechanisms that these ringed proteins utilize in binding and
translocating along the nucleic acids.
We have used presteady-state kinetics to elucidate the dynamics
of Rho protein interaction with poly(C) RNA. The kinetic pathway was
determined by monitoring the intrinsic fluorescence changes in the Rho
protein upon RNA binding using the stopped-flow method and by measuring
the presteady-state ATP hydrolysis activity. The fluorescence of Rho
protein increases when it binds to poly(C) RNA in the absence of ATP,
and the fluorescence decreases when it binds to RNA in the presence of
ATP. These fluorescence changes most likely result from global
conformational changes that affect the quantum yield of the tryptophan
or both the tryptophan and the tyrosine residues in the Rho protein,
and they provide the necessary signals to monitor in real time the
kinetics of RNA binding.
The transient changes in the fluorescence of the Rho protein upon RNA
binding were measured by the stopped-flow method, and the resulting
kinetics at varying concentrations of RNA were fit to a kinetic model
using the program Scientist. Best global fits of the fluorescence
stopped-flow kinetic data, with and without ATP, were obtained to a
three-step sequential mechanism, shown in Table I. The presteady-state
ATPase kinetics however indicated the presence of a fourth slower step
in the mechanism. This last step was necessary for the formation of a
Rho-RNA species that was competent in catalyzing ATPase turnover at the
RNA-stimulated level. The four-step mechanism shown in Table II is
therefore consistent with both the fluorescence stopped-flow and the
presteady-state ATPase kinetics. The four-step sequential mechanism
illustrated in Fig. 6 indicates that the poly(C) RNA binds to the Rho
hexamer, both with and without ATP, at a diffusion-limited rate
(k1 = 8.6 × 108
M
1 s
1)
and the resulting complex PR1 dissociates with a rate,
k
1 = 23.2 s
1. The
PR1 complex has a dissociation constant of about 30 nM, but
it is a transient species that isomerizes to PR2 with a forward rate of
k2 = 20.8 s
1 and a
reverse rate of k
2 = 1.60 s
1. The nucleotide, ATP, does not play a
significant role during the formation of PR1 and PR2. The conversion of
PR2 to PR3 is, however, stimulated by ATP. In the presence of ATP, the
PR2 intermediate isomerizes to PR3 with a forward rate,
k3, of 31.6 s
1 and
reverse rate, k
3, of 1.02 s
1. In the absence of ATP, the PR2 is
converted to PR3 at a rate of 5.0 s
1. The PR3
then appears to irreversibly convert to PR4 at a rate, k4, of 4.1 s
1 in the
presence of ATP. The presteady-state ATPase kinetics indicated that PR4
is the only species that is capable of hydrolyzing ATP at the
RNA-stimulated level.
The presteady-state ATPase kinetics showed a burst of ATP hydrolysis,
in which three ATPs were rapidly hydrolyzed per Rho hexamer, after
adding RNA. Presumably, we start with a Rho-ATP complex, with three or
four ATPs bound per hexamer (31, 33), and it appears that Rho
hydrolyzes three ATPs rapidly upon its initial encounter with the RNA.
A similar experiment in the literature (34) done under chase conditions
(where RNA was added along with excess nonradioactive ATP that acts as
a chase) showed hydrolysis of three ATPs as well, consistent with the
above proposal. The kinetic lag following the burst of ATP hydrolysis
indicates that PR1 is not capable of turning over ATP. The kinetic
simulations indicated that the species from PR1 to PR3 were
incapable of hydrolyzing ATP at the RNA-stimulated rate. The
presteady-state ATPase kinetics provided evidence for a fourth step in
the RNA binding process, PR3 to PR4 conversion, at a relatively slow
rate close to 4 s
1. The kinetics indicate
that PR4 is the only species that is competent in carrying out multiple
ATP hydrolysis events. The hydrolysis of three ATPs at a rapid rate
therefore may be a peculiarity of the initiation process. If ATP
hydrolysis is coupled to translocation, then this study suggests that
PR4 is the only species capable of translocating along RNA.
Previous studies have shown that the Rho hexamer contains two classes
of nucleic acid binding sites, the primary and secondary binding sites
(10, 11). The primary site binds pyrimidine-rich RNA or DNA with a high
affinity (13). The atomic structure of the N-terminal RNA-binding
domain consisting of the primary binding sites has been solved, and it
was suggested that the primary sites crown the hexamer (13, 14).
Electron microscopy studies of the Rho hexamer in the presence of a
tRNA showed that it binds to the outer periphery and probably to the
primary sites. Upon binding to the tRNA, the Rho hexamer showed
significant deviation from the 6-fold symmetry, with "notched" or
open rings (2). These studies suggest that the primary RNA binding
sites face outward in the hexameric ring, and when the RNA binds to
these sites, the open form of the Rho hexamer is stabilized. On the other hand, the secondary sites have been shown to bind nucleic acids
weakly, and RNA binding to these sites is allosterically coupled to ATP
binding and hydrolysis (9, 11). The secondary sites also prefer C-rich
RNA (15), and several studies indicate that these sites lie within the
central channel of the Rho ring (12, 17).
The fluorescence stopped-flow and the presteady-state ATPase kinetics
have provided a sequential RNA binding mechanism that is illustrated in
Fig. 6. The rate constants were accurately determined by global
fitting, and available results have been considered in proposing the
structures of the intermediate complexes (2, 16, 32), including the
"tethered-tracking" model (8). Our model (Fig. 6) involves the
following steps: (a) RNA binding on the outside of the ring;
(b) the wrapping of the RNA in the continuous clefts of the
primary RNA-binding sites that crown the hexamer ring; (c)
the passage of the RNA into the central channel of the ring by the
formation of a "notch" within the six-membered ring; and
(d) the activation of the coupled ATPase-translocation (5'
3') within the central channel.
The initial encounter of the RNA with the Rho hexamer occurs at a
diffusion-limited rate constant, and we have observed that this binding
is independent of ATP. Because the secondary RNA binding sites in the
central channel would not be easily accessible, we propose that the
initial diffusion-limited encounter of the RNA take place with few of
the primary RNA-binding sites resulting in PR1. Because the formation
of PR2 is also independent of ATP, we suggest that the RNA wraps around
the continuous RNA binding clefts that crown the hexamer ring during
the formation of PR2. In the presence of ATP, the PR2 isomerizes to PR3
at a relatively fast rate, and we propose that during this step the RNA
migrates into the central channel and interacts with the secondary RNA binding sites. It is very likely that the RNA retains its interaction with the primary site even after it contacts the secondary site (as
proposed by the tethered-tracking model). The last step may represent
the closing of the ring around the RNA resulting in PR4, which is the
species competent in turning over ATP, coupled to translocation.
The proposed ring-opening mechanism is consistent with the model in
which the 3'-end of the RNA from the initial attachment site passes
through the central channel in the hexamer ring (16). One possible
mechanism that does not require the ring opening would be the threading
model. In the threading mechanism, none of the subunit interfaces would
be disrupted, and a free end of the RNA would thread into the hole.
However, this mechanism is unlikely because Rho does not bind very
tightly to an RNA lacking the rut site (35), and threading
would be obstructed by the presence of the translating ribosomes on the
nascent mRNA. Hence, the ring opening is likely to be a preferred
pathway for loading hexameric Rho on the RNA under cellular conditions.
A general problem for ring proteins that bind nucleic acids in
the central channel is coordinating the events of ring opening and
nucleic acid binding in the central channel. In Rho protein, the RNA
first binds to the primary site, which we propose acts as a Rho-loading
site, and the prebound RNA is then transferred into the central channel
upon ring opening. Such a model provides a mechanism for both
sequence-specific loading of Rho on the RNA and for increasing the
efficiency of RNA binding in the central channel. This mechanism
appears to be general in the hexameric helicases that form a ring. The
bacteriophage T7 gp4 has been proposed to use the primase site as a
loading site to allow efficient binding to ssDNA (36). Consistent with
this hypothesis, the hexameric E. coli DnaB, which does not
contain a loading site within its polypeptide, binds single-stranded
DNA nearly 1000-fold more slowly than the rate observed for Rho and T7
gp4 (22). The DnaB in fact interacts with accessory proteins, such as
the DnaC protein, which loads DnaB at the replication origin
(37). It is therefore very likely that DnaC will kinetically facilitate the loading of DnaB on the DNA. The hexameric helicases such as the
SV40 large T antigen and BPV E1 have distinct origin-binding sites that
may be the loading sites for binding at the specific origins of
replication (38, 39). Phage T4 gp41 helicase associates with gp59 to
load onto T4 gp32-coated DNA (40). The MCM helicases from eukaryotes
and archaea interact with proteins such as Cdc6p that help load
them at the replication origin (41, 42). The various accessory proteins
therefore act as loading sites for helicases (21). The hexameric
helicases appear to use a general mechanism for nucleic acid binding.
The helicases form a preassociation complex that brings the DNA or RNA
in the vicinity of the central channel and increases the efficiency and
specificity of loading the nucleic acid into the central channel.