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• W2070394525 abstract "The NS3 helicase from hepatitis C virus is a prototypical DEx(H/D) RNA helicase. NS3 has been shown to unwind RNA in a discontinuous manner, pausing after long apparent steps of unwinding. We systematically examined the effects of duplex stability and ionic conditions on the periodicity of the NS3 unwinding cycle. The kinetic step size for NS3 unwinding was examined on diverse substrate sequences. The kinetic step size (16 bp/step) was found to be independent of RNA duplex stability and composition, but it exhibited strong dependence on monovalent salt concentration, decreasing to ∼11 bp/step at low [NaCl]. We addressed this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas only NS3 oligomers are capable of processive unwinding, we found that monomeric NS3 is an active helicase that unwinds with low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3, which is likely to account for the reduction in apparent step size under low salt conditions. Based on results reported here, as well as available structural and single molecule data, we present an unwinding mechanism that addresses the apparent periodicity of NS3 unwinding, the magnitude of the step size, and that integrates the various stepwise motions observed for NS3. We propose that the large kinetic step size of NS3 unwinding reflects a delayed, periodic release of the separated RNA product strand from a secondary binding site that is located in the NTPase domain (Domain II) of NS3. These findings suggest that the mechanism of product release represents an important and unexplored feature of helicase mechanism. The NS3 helicase from hepatitis C virus is a prototypical DEx(H/D) RNA helicase. NS3 has been shown to unwind RNA in a discontinuous manner, pausing after long apparent steps of unwinding. We systematically examined the effects of duplex stability and ionic conditions on the periodicity of the NS3 unwinding cycle. The kinetic step size for NS3 unwinding was examined on diverse substrate sequences. The kinetic step size (16 bp/step) was found to be independent of RNA duplex stability and composition, but it exhibited strong dependence on monovalent salt concentration, decreasing to ∼11 bp/step at low [NaCl]. We addressed this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas only NS3 oligomers are capable of processive unwinding, we found that monomeric NS3 is an active helicase that unwinds with low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3, which is likely to account for the reduction in apparent step size under low salt conditions. Based on results reported here, as well as available structural and single molecule data, we present an unwinding mechanism that addresses the apparent periodicity of NS3 unwinding, the magnitude of the step size, and that integrates the various stepwise motions observed for NS3. We propose that the large kinetic step size of NS3 unwinding reflects a delayed, periodic release of the separated RNA product strand from a secondary binding site that is located in the NTPase domain (Domain II) of NS3. These findings suggest that the mechanism of product release represents an important and unexplored feature of helicase mechanism. The DEx(H/D) proteins represent a large and ubiquitous family of putative RNA helicases (1Gorbalenya A. Koonin E.V. Curr. Opin. Struct. Biol.. 1993; 3: 419-429Google Scholar, 2Pyle A.M. Annu. Rev. Biophys.. 2008; 37: 317-336Google Scholar). The members of this highly conserved protein family are present in the vast majority of organisms, from viruses to humans, and are involved in virtually every known aspect of RNA metabolism (3Jankowsky E. Jankowsky A. Nucleic Acids Res.. 2000; 28: 333-334Google Scholar). Despite the importance of these proteins, only a small fraction of them has been well characterized, and the mechanism of their action is poorly understood. Although they are usually called “RNA helicases” because of the NTP-dependent RNA unwinding activity displayed by many DEx(H/D) proteins in vitro, other activities, such as ribonucleoprotein remodeling and RNA strand annealing, have been described for several proteins of the family, and these activities may be relevant to their functions in vivo (2Pyle A.M. Annu. Rev. Biophys.. 2008; 37: 317-336Google Scholar, 4Yang Q. Jankowsky E. Biochemistry.. 2005; 44: 13591-13601Google Scholar, 5Jankowsky E. Gross C.H. Shuman S. Pyle A.M. Science.. 2001; 291: 121-125Google Scholar, 6Fairman M.E. Maroney P.A. Wang W. Bowers H.A. Gollnick P. Nilsen T.W. Jankowsky E. Science.. 2004; 304: 730-734Google Scholar). The physiological functions and targets of most DEx(H/D) proteins remain unknown.The nonstructural protein 3 (NS3) from hepatitis C virus (HCV) 3The abbreviations used are: HCV, hepatitis C virus; MOPS, 4-morpholinepropanesulfonic acid; nt, nucleotide; RNS, randomly nicked substrate; ssRNA, single-stranded RNA. 3The abbreviations used are: HCV, hepatitis C virus; MOPS, 4-morpholinepropanesulfonic acid; nt, nucleotide; RNS, randomly nicked substrate; ssRNA, single-stranded RNA. possesses robust RNA and DNA unwinding activities and is a prototypical member of the DEx(H/D) family of ATPase proteins (7Pang P.S. Jankowsky E. Planet P.J. Pyle A.M. EMBO J.. 2002; 21: 1168-1176Google Scholar). NS3 is an essential component of the HCV replication machinery, and it is an important drug target for anti-HCV therapy. NS3 is one of the most exhaustively studied RNA helicases, and a wealth of structural and biochemical information is available for this enzyme (8Kim J.L. Morgenstern K.A. Griffith J.P. Dwyer M.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure (Lond.).. 1998; 6: 89-100Google Scholar, 9Yao N. Hesson T. Cable M. Hong Z. Kwong A.D. Le H.V. Weber P.C. Nat. Struct. Biol.. 1997; 4: 463-467Google Scholar, 10Cho H.S. Ha N.C. Kang L.W. Chung K.M. Back S.H. Jang S.K. Oh B.H. J. Biol. Chem.. 1998; 273: 15045-15052Google Scholar). Ensemble and single molecule studies have established that NS3 is a processive helicase, capable of making multiple unwinding steps of well defined size without dissociating from the substrate (11Dumont S. Cheng W. Serebrov V. Beran R.K. Tinoco Jr., I. Pyle A.M. Bustamante C. Nature.. 2006; 439: 105-108Google Scholar, 12Serebrov V. Pyle A.M. Nature.. 2004; 430: 476-480Google Scholar). This stepping behavior involves alternating pauses and rapid unwinding events and is similar to the behavior displayed by cytoskeletal motor proteins. Despite these important findings, the molecular mechanism of NS3 unwinding remains largely obscure. The pauses are likely to be caused by the necessity to reset the conformation of the helicase-RNA complex after each unwinding step, consistent with an inch-worm model of unwinding. However, the mechanism by which pauses are triggered and the role of ATP in this process are not understood. The number of base pairs unwound by NS3 per step appears to be one of the largest reported for helicases to date, and it significantly exceeds the footprint of a monomeric NS3 bound to an RNA substrate (6-8 nt) (8Kim J.L. Morgenstern K.A. Griffith J.P. Dwyer M.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure (Lond.).. 1998; 6: 89-100Google Scholar). The structural basis for these large steps is unknown, although it has been suggested that it may be related to the oligomeric state of NS3 in its active form (12Serebrov V. Pyle A.M. Nature.. 2004; 430: 476-480Google Scholar).Kinetic methods have long been employed to study helicase activity, and they provide essential tools for dissecting the mechanism of helicase function (13Ali J.A. Lohman T.M. Science.. 1997; 275: 377-380Google Scholar, 14Jankowsky E. Gross C.H. Shuman S. Pyle A.M. Nature.. 2000; 403: 447-451Google Scholar, 15Levin M.K. Wang Y.H. Patel S.S. J. Biol. Chem.. 2004; 279: 26005-26012Google Scholar, 16Lucius A.L. Vindigni A. Gregorian R. Ali J.A. Taylor A.F. Smith G.R. Lohman T.M. J. Mol. Biol.. 2002; 324: 409-428Google Scholar, 17Lohman T.M. Bjornson K.P. Annu. Rev. Biochem.. 1996; 65: 169-214Google Scholar). One of the fundamental parameters that can be measured is the kinetic step size, which is defined as the number of base pairs unwound per rate-limiting step. The kinetic step size is often reflected in a periodic mode of unwinding that is commonly displayed by processive helicases that make multiple repetitive unwinding steps before dissociating from the nucleic acid substrate (13Ali J.A. Lohman T.M. Science.. 1997; 275: 377-380Google Scholar).To characterize the mechanism of RNA unwinding by NS3 and to develop a physical explanation for its unusual stepping behavior, we systematically studied the dependence of the NS3 kinetic step size on duplex stability and ionic conditions. We find that the step size of NS3 shows no dependence on duplex stability or sequence, in contrast to its processivity and unwinding rate constant, which have been previously shown to strongly depend on duplex stability (18Cheng W. Dumont S. Tinoco Jr., I. Bustamante C. Proc. Natl. Acad. Sci. U. S. A.. 2007; 104: 13954-13959Google Scholar, 19Donmez I. Rajagopal V. Jeong Y.J. Patel S.S. J. Biol. Chem.. 2007; 282: 21116-21123Google Scholar). However, we observe that the apparent kinetic step size of RNA unwinding by NS3 is sensitive to monovalent salt concentration and is reduced to ∼11 bp under low salt conditions. We address this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas NS3 oligomers are capable of processive unwinding, we find that monomeric NS3 is an active helicase that possesses low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3 and are likely to account for the changes in kinetic step size of NS3 under low salt conditions. Combining these results with data from previous studies, we propose a physical unwinding mechanism that explains the kinetic step size and the diverse kinetic behaviors that are displayed by NS3.EXPERIMENTAL PROCEDURESMaterials—DNA oligonucleotides were obtained from Keck Facility, Yale University. Buffers, salts, and ATP were from Sigma. Phosphorothioate NTPs were purchased from TriLink Biotechnologies. Full-length NS3 (genotype 1a) was expressed and purified as described (20Beran R.K. Bruno M.M. Bowers H.A. Jankowsky E. Pyle A.M. J. Mol. Biol.. 2006; 358: 974-982Google Scholar). The expressed protein contained a tag of following sequence at its N terminus, MRGSHHHHHHGSDYKDDDDKA. This particular tag, which greatly facilitates NS3 purification, does not influence unwinding step size or functional complex formation characteristics when compared with untagged protein (data not shown). 4V. Serebrov and R. K. F. Beran, unpublished information. Preparation of RNS Duplexes—RNA strands were prepared by T7 transcription of PCR-generated DNA templates using the MEGAshortscript RNA transcription kit (Ambion). For each substrate, the top strand RNA (Fig. 2) was synthesized in four separate transcription reactions. Each of the reactions contained one of the α-phosphorothioate NTPs along with the four regular NTPs. To achieve a suitable level of phosphorothioate incorporation (∼0.7 nicks per top strand upon iodine cleavage), the concentration of each α-phosphorothioate NTP was equal to that of the corresponding NTP (3.5 mm). After transcription, the four RNA products were purified on denaturing PAGE and combined in equimolar proportions to yield a pool of top strand RNAs containing phosphorothioate linkages at random positions. The bottom strands were unmodified RNAs.To prepare RNS duplexes, the pool of top strand RNS oligonucleotides was 5′-end-labeled with [γ-32P]ATP (PerkinElmer Life Sciences) using T4 polynucleotide kinase (New England Biolabs) and annealed with 2× molar excess of unlabeled complementary bottom strand in the annealing buffer (20 mm MOPS-NaOH, pH 6.5, 30 mm NaCl and 1 mm EDTA) by heating to 95 °C and then cooling to room temperature over 30 min. The phosphorothioate linkages in the top strands were then cleaved by addition of iodine to 0.1 mm final concentration and incubating for 15 min at room temperature. The cleaved substrates were then purified by electrophoresis on a semi-denaturing polyacrylamide gel (15% acrylamide, 3 m urea, and 0.5× Tris borate electrophoresis buffer, TBE).NS3 Unwinding Kinetics—Quench-flow unwinding experiments were performed using a model RQF-3 KinTek quench-flow apparatus maintained at 37 °C by a circulating water bath. Prior to initiation of unwinding, the radiolabeled RNS duplexes (2 nm) were preincubated with 200 nm NS3 in the standard reaction buffer (20 mm MOPS-NaOH, pH 6.5, 30 mm NaCl, 3 mm MgCl2, 2 mm dithiothreitol, 1% glycerol, 0.2% Triton X-100) for 1 h at 37 °C. Unwinding reactions were initiated in the quench-flow apparatus by mixing the preincubated reaction mix with an equal volume of 8 mm ATP and 4 μm trap oligonucleotide in the standard reaction buffer. The oligonucleotide used as a trap for NS3 was a 60-mer DNA with the sequence CATATGAGTCGTATCGTG(T)42. The unwinding reactions were quenched at various times (0.005 to 5 s) with another volume of quench buffer containing 30 mm EDTA, 25% sucrose, 1% SDS, 0.015% xylene cyanol, and 0.015% bromphenol blue. The addition of EDTA and SDS causes NS3 to dissociate from the RNA. Because all partially unwound duplexes immediately reassociate upon NS3 dissociation, only those product lengths that were completely unwound before quenching of the reaction were detected. The quenched reactions were analyzed by electrophoresis on a semi-denaturing polyacrylamide gel (15% acrylamide, 3 m urea, 0.5× TBE buffer). No detectable spontaneous duplex dissociation occurred in the gel under these conditions. The electrophoretic bands were visualized and quantified using a Storm 840 PhosphorImager and Image-Quant software (GE Healthcare). The fraction unwound was determined for each time point and for each RNS duplex length using the following formula: Aunw = Iunw/Iheat, where Iunw and Iheat are relative intensities of electrophoretic bands corresponding to the unwinding reaction and heat-denatured substrate, respectively, corrected for total amounts of radioactivity loaded onto the respective gel lanes.Analysis of Unwinding Time Courses—Individual time courses of NS3 unwinding were analyzed using the analytical solution for Scheme 1 obtained through Laplace transform (37Lucius A.L. Maluf N.K. Fischer C.J. Lohman T.M. Biophys. J.. 2003; 85: 2224-2239Google Scholar) and continuous in both k and n as shown in Equation 1, At(t)=A⋅Γ(kt, n)(Eq. 1) where At(t) is the fraction unwound; t is unwinding time; A is the maximal unwinding amplitude, and Γ(kt, n) is incomplete γ function, defined as shown in Equation 2,The fitting of time courses to Equation 1 was performed using a custom-written script for MATLAB 7.0. This script will be made available at Pyle laboratory website. Fitting was performed individually for each unwinding time course. All unwinding parameters (unwinding amplitude, kinetic rate constant, and number of steps) were allowed to float.RESULTSTo determine the kinetic step size of NS3 under diverse reaction conditions, we conducted unwinding experiments with a combinatorial RNA library of randomly nicked substrates, using methodologies that were previously developed in our laboratory (12Serebrov V. Pyle A.M. Nature.. 2004; 430: 476-480Google Scholar). This approach takes advantage of the fact that NS3 translocates along the “bottom strand” of the duplex and readily displaces “top strand” sequences that contain lesions or discontinuities (Fig. 1) (20Beran R.K. Bruno M.M. Bowers H.A. Jankowsky E. Pyle A.M. J. Mol. Biol.. 2006; 358: 974-982Google Scholar). During the course of unwinding, when the helicase passes a nick, the unwound 5′-portion of the top strand is released, thereby reporting on the progress of helicase translocation and duplex unwinding at a particular location and time. We have employed this approach to examine intermediate states of unwinding using a pool of RNA substrates that contain nicks at random positions in the top strand.FIGURE 1General scheme for the RNS unwinding experiments. The pool of substrates with randomly nicked top strand is created by annealing the 5′-labeled top strand RNA containing randomly incorporated phosphorothioate linkages (less than 1 incorporation per top strand molecule) with the complementary bottom strand, followed by iodine cleavage. NS3 is prebound to the single-stranded overhangs of the substrate molecules in the absence of ATP, and then the synchronized unwinding reaction is initiated by ATP addition. The radioactively labeled 5′-terminal portions of the top strand are released when unwinding of that region is complete and they are subsequently analyzed by gel electrophoresis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The nicks are generated by “doping” the top strand with phosphorothioate linkages during the T7 transcription reaction. The top strand RNAs containing phosphorothioates are then 5′-end-labeled with 32P and annealed to the bottom strands, and the phosphorothioate linkages are cleaved by iodine. The resulting pool of randomly nicked substrates is then subjected to an unwinding reaction, and the resultant products are resolved by gel electrophoresis. Because the nick positions are completely random, every possible nucleotide position along the top strand is represented by a fraction of molecules in the substrate pool, and the amount of the unwound product can be independently determined for all the RNS lengths by quantitating the corresponding bands on the gel.To address the influence of sequence on NS3 unwinding parameters, we synthesized four different randomly nicked RNA substrates (Fig. 2). Each of the substrates consists of an RNA duplex that is flanked by a single-stranded 18-nt 3′-overhang that is required for NS3 binding and initiation of unwinding. The overhang sequence was identical for all of the substrates. The length of the duplex part was either 80 (RAND1 and RAND2 substrates) or 82 bp (FLAT1 and FLAT2), to allow for multiple unwinding steps by NS3. The four RNA duplexes used in this study were designed to explore NS3 behavior in a diverse set of environments, with variations in GC content and stability profiles. RAND 1 and RAND2 are substrates of random sequence and average GC contents of 60.0 and 45.0%, respectively. The substrate FLAT1 is composed of the repeating tetranucleotide sequence ACTG, flanked with short irregular sequences (Fig. 2, underlined letters) to ensure proper annealing of strands. The repeat sequence was selected so that the variations of free energy for base pairs within the repeat were minimal, based on the nearest-neighbor rules (21Freier S.M. Kierzek R. Jaeger J.A. Sugimoto N. Caruthers M.H. Neilson T. Turner D.H. Proc. Natl. Acad. Sci. U. S. A.. 1986; 83: 9373-9377Google Scholar). This results in nearly uniform thermodynamic stability for the repetitive portion of the substrate duplex. The “flat” profile of thermodynamic stability essentially eliminates the idiosyncrasy originating from “rugged” energetic profiles of substrates with random sequences, thereby providing an important control for unwinding behavior that otherwise might be obscured by energetic irregularities of the duplex. Finally, the substrate FLAT2 is identical to FLAT1, with the exception of 7 bp in the middle of the duplex that have been changed to GCCCCCC (Fig. 2, boxed sequence, nucleotides 37-43). This creates a short stretch of RNA duplex that is considerably more stable than the surrounding sequence and has the potential to promote altered unwinding behavior of NS3 (e.g. cause changes in its unwinding periodicity) because of the sudden increase in its “workload.”We carried out time-resolved NS3 unwinding experiments with the four RNS duplexes under identical conditions (37 °C, 4 mm ATP), using a rapid quench-flow mixer. The unwinding reactions were quenched at various times (0.005 to 5 s), and the unwinding products were resolved on a semi-denaturing polyacrylamide gel (Fig. 3A). Each experiment resulted in a series of 30-40 unwinding time courses for various product lengths, all of which could be visualized with single nucleotide resolution (Fig. 3B). With the exception of the shortest resolved RNS lengths, all kinetic series exhibited delayed unwinding kinetics (manifested as lag phases in the unwinding time courses), indicating the presence of rate-limiting intermediate states of unwinding.FIGURE 3A, representative polyacrylamide gel showing separation of the unwinding products after performing a time-resolved RNS experiment with substrate FLAT1. The unwinding reactions were initiated by addition of ATP and terminated at various times (0.005 to 5 s) using a quench-flow mixer, and analyzed on a semi-native polyacrylamide gel as described under “Experimental Procedures.” The last two lanes on the right are the RNS substrate heated at 99 °C for 5 min (>95% strand dissociation) and a sequencing ladder, respectively. B, kinetic time courses obtained by quantitating the autoradiogram of the gel shown in A. Product lengths up to ∼50 bp can be resolved with single nucleotide resolution.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Unwinding time courses were analyzed using the n-step kinetic Scheme 1 (13Ali J.A. Lohman T.M. Science.. 1997; 275: 377-380Google Scholar),where n represents the number of rate-limiting steps, and n-1 is the number of populated intermediate states of unwinding (I1, I2,···, In-1). S and P correspond to the substrate and final product, respectively, and k is the kinetic rate constant of each step. Fitting of data to Scheme 1 was performed using Equation 1 as described under “Experimental Procedures.” For each RNA substrate, the numbers of rate-limiting kinetic steps determined from the NLLS procedure were plotted versus corresponding RNS lengths (Fig. 4). The average kinetic step sizes (bp/step) and associated standard deviations were then determined by linear regression (Table 1). The unwinding rate constants were found to be independent of RNS duplex length within our experimental uncertainty (Table 1). Despite considerable differences in the thermodynamic stabilities of our substrates, we observed an identical unwinding step size (16 ± 1 bp/step) for all of the substrates under our standard unwinding conditions (20 mm MOPS-NAOH, pH 6.5, 30 mm NaCl). This number provides a more precise estimate than that which was previously reported (18 ± 2 bp/step) (12Serebrov V. Pyle A.M. Nature.. 2004; 430: 476-480Google Scholar).FIGURE 4The dependence of the number of rate-limiting steps on RNS duplex length, determined from the analysis of experimental unwinding time courses for each of the RNS substrates. Data for different substrates are shown in different colors as follows: black (RAND1), blue (RAND2), red (FLAT1), and green (FLAT2). The numbers of rate-limiting unwinding steps (symbols) were determined from unwinding time courses by performing the NLLS fitting to Equation 1 as described under “Experimental Procedures.” Solid lines represent linear fits.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Kinetic parameters of NS3 unwinding on four RNA substratesSubstrateStep size, bp/stepUnwinding rate constant, steps/sRAND116.3 ± 1.11.1 ± 0.3RAND216.5 ± 1.00.9 ± 0.2FLAT116.3 ± 1.11.3 ± 0.2FLAT215.9 ± 1.01.5 ± 0.3Average16.3 ± 0.31.2 ± 0.3 Open table in a new tab Kinetic Step Size of NS3 Unwinding Is Sensitive to Monovalent Salt Concentration—It was previously proposed that the large kinetic step size exhibited by NS3 in bulk is the consequence of NS3 dimerization on the RNA substrate (12Serebrov V. Pyle A.M. Nature.. 2004; 430: 476-480Google Scholar). However, additional insights were provided by high resolution single molecule studies using optical tweezers (11Dumont S. Cheng W. Serebrov V. Beran R.K. Tinoco Jr., I. Pyle A.M. Bustamante C. Nature.. 2006; 439: 105-108Google Scholar, 18Cheng W. Dumont S. Tinoco Jr., I. Bustamante C. Proc. Natl. Acad. Sci. U. S. A.. 2007; 104: 13954-13959Google Scholar). These experiments demonstrated that, under conditions of relatively high pulling force, NS3 unwinds RNA duplexes with a somewhat smaller step size of 11 ± 3 bp, which was attributed to unwinding by a monomeric form of NS3. Significantly, NS3 requires relatively high pulling forces to behave as a processive helicase in the optical trap.To better understand the relationship between step size, oligomerization state, NS3 affinity, and structural integrity of the duplex, we examined the kinetic step size for NS3 under different monovalent salt conditions. RNS unwinding experiments were conducted in a reaction buffer that contained varying concentrations of NaCl (from 0 to 100 mm, in addition to 20 mm MOPS-NaOH, pH 6.5). The numbers of unwinding steps for different RNS duplex lengths were determined by fitting the resultant time courses to Equation 1 for substrate FLAT1 under various salt concentrations (Fig. 5A). The kinetic step sizes at different salt concentrations were determined from the slope of the linear fit to the data, revealing a substantial dependence of step size on monovalent salt concentration (Fig. 5B). Indeed, under conditions of lowest ionic strength, the NS3 step size reduces to 11 bp, which is exactly the value observed in the optical tweezer experiments.FIGURE 5The apparent kinetic step size of RNA unwinding by NS3 is sensitive to monovalent salt concentration. A, numbers of kinetic steps determined for the substrate FLAT1 at 0, 30, and 60 mm NaCl (in addition to 20 mm MOPS-NaOH, pH 6.5) plotted versus RNS duplex length. B, apparent step size of NS3 unwinding at varying NaCl concentrations determined from the slopes of linear fits from a. Except for 100 mm NaCl, duplicate experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Determination of the Functional Oligomeric State of NS3 on RNA Substrates—We set out to determine whether the oligomeric state of NS3 in the functional RNA complex can be modulated by salt concentration and thereby account for the observed changes of the unwinding step size. To study the kinetics of functional complex formation, we performed “double mixing” experiments (Fig. 6), in which the single cycle reaction amplitude of NS3 unwinding (Aunw) is measured as a function of time of preincubation of NS3 with RNA substrate. It has been established that, under single cycle conditions, Aunw is dependent on both functional complex concentration and RNA duplex length (13Ali J.A. Lohman T.M. Science.. 1997; 275: 377-380Google Scholar) as shown in Equation 3, Γ(kt, n)≡∫0kttn−1e−1dtI∫0∞tn−1e−1dt(Eq. 2) where [EnS*] is concentration of NS3-RNA functional complex; p is processivity per unwinding step; L is the duplex length, and s is unwinding step size.FIGURE 6Top, the general scheme of formation of functional NS3-RNA complex, where S is an RNA substrate; E is NS3 protein; P is unwound product; EnS* is the functional complex, and n is number of NS3 molecules bound to the RNA substrate in the functional complex. Bottom, the experimental design of functional complex formation experiments. The RNA substrate is preincubated with NS3 for variable time Δt, after which unwinding is initiated by the addition of ATP and trap oligonucleotide. The trap oligonucleotide is included to sequester any unbound protein and to prevent re-binding of NS3 to the RNA substrate during the unwinding reaction (single cycle unwinding conditions). The unwinding reactions are allowed to proceed to completion (90 s), after which unwinding amplitudes (Aunw) are determined for each Δt by resolving the unwound products by native PAGE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Equation 3 describes the relationship between the measured amount of unwound product and the concentration of the functional complex. One important implication of Equation 3 is that if observed unwinding is produced by a homogeneous population of active NS3 species, then the kinetics of functional complex formation measured with different RNA duplex lengths should be identical and differ only in their amplitudes. However, if unwinding results from a heterogeneous mixture of functionally active species (e.g. different oligomeric states of NS3), and at least some of these species have substantially different levels of processivity, then different kinetics of functional complex formation can be observed for different RNA duplex lengths. For instance, if nonprocessive species are present, they will only contribute to kinetics of functional complex formation measured with very short RNA duplexes. Measuring functional complex formation kinetics with duplexes of variable length can therefore enable one to resolve different active species of NS3.To establish if NS3 unwinding can be at" @default.
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• W2070394525 title "Establishing a Mechanistic Basis for the Large Kinetic Steps of the NS3 Helicase" @default.
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