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• W1569649547 abstract "The Escherichia coli transcription termination factor Rho is structurally and functionally homologous to hexameric helicases that assemble into ring structures. Using stopped-flow fluorescence and presteady-state ATPase kinetics, we have determined the kinetic pathway of poly(C) RNA binding to Rho hexamer, both in the presence and in absence of ATP. These studies indicate a four-step sequential mechanism of RNA binding and reveal the respective roles of the primary and secondary RNA binding sites in initiation and ATPase activation of Rho. The primary RNA binding sites of Rho hexamer interact with poly(C) RNA at a diffusion-limited rate constant close to 8 × 108m−1s−1, resulting in the Rho-RNA species PR1, which subsequently isomerizes to PR2 with a rate constant 21 s−1. The PR2 isomerizes to PR3 with a rate constant of 32 s−1 in the presence of ATP, and the formation of PR4 from PR3 results in a species that is fully competent in hydrolyzing ATP at the RNA-stimulated rate. The PR3 to PR4 isomerization occurs at a relatively slow rate of 4.1 s−1; thus, the presteady-state ATPase kinetics show a distinct lag due to the slow initiation step. The interactions of the RNA with the primary sites trigger ring opening, and we propose that during the last two steps, the RNA migrates into the central channel and interacts with the secondary sites, resulting in the activation of the ATPase activity. The primary RNA binding sites, in addition to promoting sequence specific initiation, kinetically facilitate loading of the RNA into the secondary sites, which are relatively inaccessible, since they are present in the central channel. These studies reveal common features used by hexameric helicases to bind nucleic acids in an efficient and specific manner. The Escherichia coli transcription termination factor Rho is structurally and functionally homologous to hexameric helicases that assemble into ring structures. Using stopped-flow fluorescence and presteady-state ATPase kinetics, we have determined the kinetic pathway of poly(C) RNA binding to Rho hexamer, both in the presence and in absence of ATP. These studies indicate a four-step sequential mechanism of RNA binding and reveal the respective roles of the primary and secondary RNA binding sites in initiation and ATPase activation of Rho. The primary RNA binding sites of Rho hexamer interact with poly(C) RNA at a diffusion-limited rate constant close to 8 × 108m−1s−1, resulting in the Rho-RNA species PR1, which subsequently isomerizes to PR2 with a rate constant 21 s−1. The PR2 isomerizes to PR3 with a rate constant of 32 s−1 in the presence of ATP, and the formation of PR4 from PR3 results in a species that is fully competent in hydrolyzing ATP at the RNA-stimulated rate. The PR3 to PR4 isomerization occurs at a relatively slow rate of 4.1 s−1; thus, the presteady-state ATPase kinetics show a distinct lag due to the slow initiation step. The interactions of the RNA with the primary sites trigger ring opening, and we propose that during the last two steps, the RNA migrates into the central channel and interacts with the secondary sites, resulting in the activation of the ATPase activity. The primary RNA binding sites, in addition to promoting sequence specific initiation, kinetically facilitate loading of the RNA into the secondary sites, which are relatively inaccessible, since they are present in the central channel. These studies reveal common features used by hexameric helicases to bind nucleic acids in an efficient and specific manner. The Escherichia coli Rho protein is a transcription termination factor that assembles into a hexamer of six identical protein subunits arranged in a ring (1Gogol E.P. Seifried S.E. von Hippel P.H. J. Mol. Biol... 1991; 221: 1127-1138Google Scholar, 2Yu X. Horiguchi T. Shigesada K. Egelman E.H. J. Mol. Biol... 2000; 299: 1279-1287Google Scholar). 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 (3Roberts J.W. Nature.. 1969; 223: 480-482Google Scholar, 4Geiselmann J. Wang Y. Seifried S.E., von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A... 1993; 90: 7754-7758Google Scholar, 5Richardson J.P. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology.American Society for Microbiology Press. 1996; : 822-848Google Scholar). 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 (6Brennan C.A. Dombroski A.J. Platt T. Cell.. 1987; 48: 945-952Google Scholar). 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 (6Brennan C.A. Dombroski A.J. Platt T. Cell.. 1987; 48: 945-952Google Scholar, 7Galluppi G.R. Richardson J.P. J. Mol. Biol... 1980; 138: 513-539Google Scholar, 8Steinmetz E.J. Platt T. Proc. Natl. Acad. Sci. U. S. A... 1994; 91: 1401-1405Google Scholar).The interactions of the Rho hexamer with the RNA are mediated by two classes of RNA binding sites (9Richardson J.P. J. Biol. Chem... 1982; 257: 5760-5766Google Scholar, 10Geiselmann J. Yager T.D. von Hippel P.H. Protein Sci... 1992; 1: 861-873Google Scholar, 11Wang Y. von Hippel P.H. J. Biol. Chem... 1993; 268: 13947-13955Google Scholar, 12Miwa Y. Horiguchi T. Shigesada K. J. Mol. Biol... 1995; 254: 815-837Google Scholar). 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 (13Bogden C.E. Fass D. Bergman N. Nichols M.D. Berger J.M. Mol. Cell.. 1999; 3: 487-493Google Scholar). These and other structural studies indicate that the primary RNA binding sites crown the Rho hexamer and are easily accessible for RNA binding (2Yu X. Horiguchi T. Shigesada K. Egelman E.H. J. Mol. Biol... 2000; 299: 1279-1287Google Scholar, 13Bogden C.E. Fass D. Bergman N. Nichols M.D. Berger J.M. Mol. Cell.. 1999; 3: 487-493Google Scholar, 14Allison T.J. Wood T.C. Briercheck D.M. Rastinejad F. Richardson J.P. Rule G.S. Nat. Struct. Biol... 1998; 5: 352-356Google Scholar). The RNA has been proposed to wrap around the primary RNA binding sites of the Rho hexamer (13Bogden C.E. Fass D. Bergman N. Nichols M.D. Berger J.M. Mol. Cell.. 1999; 3: 487-493Google Scholar, 16Richardson J.P. J. Biol. Chem... 1996; 271: 1251-1254Google Scholar), consistent with the protection of 70–80 nucleotides of poly(C) RNA (15McSwiggen J.A. Bear D.G. von Hippel P.H. J. Mol. Biol... 1988; 199: 609-622Google Scholar). Several studies indicate that the Rho hexamer contains a secondary RNA binding site that is distinct from the primary site (9Richardson J.P. J. Biol. Chem... 1982; 257: 5760-5766Google Scholar, 11Wang Y. von Hippel P.H. J. Biol. Chem... 1993; 268: 13947-13955Google Scholar, 12Miwa Y. Horiguchi T. Shigesada K. J. Mol. Biol... 1995; 254: 815-837Google Scholar, 17Pereira S. Platt T. J. Mol. Biol... 1995; 251: 30-40Google Scholar). 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 (12Miwa Y. Horiguchi T. Shigesada K. J. Mol. Biol... 1995; 254: 815-837Google Scholar). 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 (18Yu X. Hingorani M.M. Patel S.S. Egelman E.H. Nat. Struct. Biol... 1996; 3: 740-743Google Scholar, 19Bujalowski W. Jezewska M.J. Biochemistry.. 1995; 34: 8513-8519Google Scholar, 20Morris P.D. Raney K.D. Biochemistry.. 1999; 38: 5164-5171Google Scholar). In Rho, the interactions of RNA with the secondary sites are necessary for ATPase activation (9Richardson J.P. J. Biol. Chem... 1982; 257: 5760-5766Google Scholar).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 (21Ahnert P. Picha K.M. Patel S.S. EMBO J... 2000; 19: 3418-3427Google Scholar) andE. coli DnaB (22Bujalowski W. Jezewska M.J. J. Mol. Biol... 2000; 295: 831-852Google Scholar).RESULTSWe 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 (15McSwiggen J.A. Bear D.G. von Hippel P.H. J. Mol. Biol... 1988; 199: 609-622Google Scholar, 28Lowery C. Richardson J.P. J. Biol. Chem... 1977; 252: 1381-1385Google Scholar). 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 BindingThe 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.Stopped-flow Kinetics of Poly(C) Binding to Rho Hexamer in the Absence of ATPThe 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 μmpolymer) 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 (25Geiselmann J. Yager T.D. Gill S.C. Calmettes P. von Hippel P.H. Biochemistry.. 1992; 31: 111-121Google Scholar). Fig. 2 Ashows 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. 2 A. 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.Figure 2Stopped-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 × 108m−1 s−1and an intercept of 11.0 s−1. D, the slower rate constant (k2) increased hyperbolically with RNA concentration and fit to a hyperbola (Equation3) with a K12 of 50 nm, a maximum rate of 4.7 ± 0.6 s−1, and a yintercept of 1.2 s−1.View Large Image Figure ViewerDownload (PPT)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 μmhexamer) 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.2 B. 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. 2 C). 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 × 108m−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. 2 D). 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 (29Picha K.M. Ahnert P. Patel S.S. Biochemistry.. 2000; 39: 6401-6409Google Scholar). 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 × 108m−1s−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 ofk−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.Table IThree-step mechanism of poly(C) RNA binding to E. coli Rho in the absence or presence of ATPP+R⇄k−1k1PR1⇄k−2k2PR2⇄k−3k3PR3k1k−1k2k−2k3k−3m−1s−1s−1s−1s−1s−1s−1Without ATP7.47 ± 0.14 × 10812.0 ± 0.7726.2 ± 1.532.81 ± 0.865.00 ± 0.46<0.02With ATP8.51 ± 0.13 × 10822.9 ± 1.2320.5 ± 0.771.58 ± 1.5131.2 ± 1.930.025 ± 0.013 Open table in a new tab Stopped-flow Kinetics of Poly(C) Binding to Rho Hexamer in the Presence of ATPPrevious studies have shown that ATP increases the affinity of Rho protein for RNA (30Gan E. Richardson J.P. Biochemistry.. 1999; 38: 16882-16888Google Scholar). Under equilibrium conditions, in the absence of RNA, the Rho hexamer binds three or four ATP molecules with very little hydrolysis (31Stitt B.L. J. Biol. Chem... 1988; 263: 11130-11137Google Scholar). 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. 3 A. 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. 3 A). 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.Figure 3Stopped-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) withk1 = 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 μmhexamer) 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 inB 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 × 108m−1s−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 K12 of 57 nm and a maximum rate of 31.5 ± 5.6 s−1.View Large Image Figure ViewerDownload (PPT)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. 3 B) 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 × 108m−1 s−1(Fig. 3 C), 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.3 D). 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. 2 D). 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. 3 B 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 × 108m−1s−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 andk−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 HydrolysisThe 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 (11Wang Y. von Hippel P.H. J. Biol. Chem... 1993; 268: 13947-13955Google Scholar, 32Burgess B.R. Richardson J.P. J. Biol. Chem... 2001; 276: 4182-4189Google Scholar). The primary RNA binding site is accessible, whereas the secondary site is proposed to lie within the central channel (12Miwa Y. Horiguchi T. Shigesada K. J. Mol. Biol... 1995; 254: 815-837Google Scholar). It is also known that the steady-state ATPase activity of Rho is stimulated only when RNA interacts with the secondary RNA-binding sites (9Richardson J.P. J. Biol. Chem... 1982; 257: 5760-5766Google Scholar). Thus, RNA needs to travel and bind within the central channel of the Rho hexamer to activate the steady-state ATPase activity (12Miwa Y. Horiguchi T. Shigesada K. J. Mol. Biol... 1995; 254: 815-837Google Scholar, 16Richardson J.P. J. Biol. Chem... 1996; 271: 1251-1254Google Scholar). 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 [α-32P]ATP for 5 or 10 s and then rapidly mixed with poly(C) RNA (Fig. 4 A). The formation of ADP after varying times of incubation with the RNA was measured, and the resulting data are shown in Fig. 4 B. 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.Figure 4The 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 [α-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 μmpolymer), 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 lineshows the predicted curve for a mechanism in which PR3 and PR4 hydrolyzed ATP at the poly(C) RNA-stimulated rate of 30 s−1.View Large Image Figure ViewerDownload (PPT)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. 4 B. 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 (TableII). 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 MechanismThe 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.5 A) 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 (2Yu X. Horiguchi T. Shigesada K. Egelman E.H. J. Mol. Biol... 2000; 299: 1279-1287Google Scholar, 32Burgess B.R. Richardson J.P. J. Biol. Chem... 2001; 276: 4182-4189Google Scholar), 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" @default.
• W1569649547 created "2016-06-24" @default.
• W1569649547 creator A5026036097 @default.
• W1569649547 creator A5034388856 @default.
• W1569649547 date "2001-04-01" @default.
• W1569649547 modified "2023-09-29" @default.
• W1569649547 title "The Kinetic Pathway of RNA Binding to the Escherichia coli Transcription Termination Factor Rho" @default.
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