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• W2055140930 abstract "We have constructed a structural model for poliovirus RNA-dependent RNA polymerase (3Dpol) in complex with a primer-template (sym/sub) and ATP. Residues found in conserved structural motifs A (Asp-238) and B (Asn-297) are involved in nucleotide selection. Asp-238 appears to couple binding of nucleotides with the correct sugar configuration to catalytic efficiency at the active site of the enzyme. Asn-297 is involved in selection of ribonucleoside triphosphates over 2′-dNTPs, a role mediated most likely via a hydrogen bond between the side chain of this residue and the 2′-OH of the ribonucleoside triphosphate. Substitutions at position 238 or 297 of 3Dpolproduced derivatives exhibiting a range of catalytic efficiencies when assayed in vitro for poly(rU) polymerase activity or sym/sub elongation activity. A direct correlation existed between activity on sym/sub and biological phenotypes; a 2.5-fold reduction in polymerase elongation rate produced virus with a temperature-sensitive growth phenotype. These data permit us to propose a detailed, structural model for nucleotide selection by 3Dpol, confirm the biological relevance of the sym/sub system, and provide additional evidence for kinetic coupling between RNA synthesis and subsequent steps in the virus life cycle. We have constructed a structural model for poliovirus RNA-dependent RNA polymerase (3Dpol) in complex with a primer-template (sym/sub) and ATP. Residues found in conserved structural motifs A (Asp-238) and B (Asn-297) are involved in nucleotide selection. Asp-238 appears to couple binding of nucleotides with the correct sugar configuration to catalytic efficiency at the active site of the enzyme. Asn-297 is involved in selection of ribonucleoside triphosphates over 2′-dNTPs, a role mediated most likely via a hydrogen bond between the side chain of this residue and the 2′-OH of the ribonucleoside triphosphate. Substitutions at position 238 or 297 of 3Dpolproduced derivatives exhibiting a range of catalytic efficiencies when assayed in vitro for poly(rU) polymerase activity or sym/sub elongation activity. A direct correlation existed between activity on sym/sub and biological phenotypes; a 2.5-fold reduction in polymerase elongation rate produced virus with a temperature-sensitive growth phenotype. These data permit us to propose a detailed, structural model for nucleotide selection by 3Dpol, confirm the biological relevance of the sym/sub system, and provide additional evidence for kinetic coupling between RNA synthesis and subsequent steps in the virus life cycle. Klenow fragment ribonucleoside triphosphate human immunodeficiency virus-1 reverse transcriptase Moloney murine leukemia virus RNA-dependent RNA polymerase DNA-dependent RNA polymerase polymerase chain reaction All nucleic acid polymerases, with the exception of mammalian DNA polymerase β, have the same overall topology (1Hansen J.L. Long A.M. Schultz S.C. Structure. 1997; 5: 1109-1122Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). As suggested first by Steitz in his description of the Klenow fragment of DNA polymerase I (KF)1 (2Ollis D.L. Kline C. Steitz T.A. Nature. 1985; 313: 818-819Crossref PubMed Scopus (88) Google Scholar), these enzymes resemble a cupped, right hand with fingers, palm, and thumb subdomains. The fingers and thumb subdomains contribute to substrate binding, especially to regions of primer and template remote from the catalytic center (3Eick D. Wedel A. Heumann H. Trends Genet. 1994; : 292-296Abstract Full Text PDF PubMed Scopus (33) Google Scholar, 4Hermann T. Meier T. Gotte M. Heumann H. Nucleic Acids Res. 1994; 22: 4625-4633Crossref PubMed Scopus (45) Google Scholar, 5Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1100) Google Scholar, 6Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (479) Google Scholar, 7Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1355) Google Scholar). The palm subdomain of all classes of polymerase contains structural elements necessary for phosphoryl transfer and binding to primer, template, and nucleotide (8Doublie S. Ellenberger T. Curr. Opin. Struct. Biol. 1998; 8: 704-712Crossref PubMed Scopus (174) Google Scholar, 9Boyer P.L. Ferris A.L. Clark P. Whitmer J. Frank P. Tantillo C. Arnold E. Hughes S.H. J. Mol. Biol. 1994; 243: 472-483Crossref PubMed Scopus (82) Google Scholar, 10Ago H. Adachi T. Yoshida A. Yamamoto M. Habuka N. Yatsunami K. Miyano M. Struct. Fold. Des. 1999; 7: 1417-1426Abstract Full Text Full Text PDF Scopus (387) Google Scholar, 11Lesburg C.A. Cable M.B. Ferrari E. Hong Z. Mannarino A.F. Weber P.C. Nat. Struct. Biol. 1999; 6: 937-943Crossref PubMed Scopus (693) Google Scholar, 12Steitz T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6498-6502Crossref PubMed Scopus (1023) Google Scholar). The overall structure and, to some extent, sequence of palm subdomains are also highly homologous. Thus, the functional similarity between the kinetic and chemical mechanism of nucleic acid polymerases is not surprising (13Steitz T.A. J. Biol. Chem. 1999; 274: 17395-17398Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar, 14Kuchta R.D. Mizrahi V. Benkovic P.A. Johnson K.A. Benkovic S.J. Biochemistry. 1987; 26: 8410-8417Crossref PubMed Scopus (338) Google Scholar, 15Patel S.S. Wong I. Johnson K.A. Biochemistry. 1991; 30: 511-525Crossref PubMed Scopus (469) Google Scholar, 16Reardon J.E. J. Biol. Chem. 1993; 268: 8743-8751Abstract Full Text PDF PubMed Google Scholar, 17Jia Y. Patel S.S. J. Biol. Chem. 1997; 272: 30147-30153Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Nucleic acid polymerases are categorized based upon their specificity for template and nucleotide. Of course, specificity is a relative term, since it is quite dependent upon reaction conditions. At physiologically relevant values of pH and ionic strength and in the presence of Mg2+ ions, most DNA-dependent DNA polymerases prefer to utilize DNA templates and 2′-deoxyribonucleotides (2′-dNTPs) as substrates rather than RNA and ribonucleotides (rNTPs) (18Benkovic S.J. Cameron C.E. Methods Enzymol. 1995; 262: 257-269Crossref PubMed Scopus (48) Google Scholar). The converse is true for RNA-dependent RNA polymerases (RdRPs) (19Arnold J.J. Cameron C.E. J. Biol. Chem. 1999; 274: 2706-2716Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 20Arnold J.J. Ghosh S.K. Cameron C.E. J. Biol. Chem. 1999; 274: 37060-37069Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, even under physiological conditions, exceptions to polymerase specificity have been noted, especially for primer and/or template utilization. For example, KF utilizes RNA templates (21Ricchetti M. Buc H. EMBO J. 1993; 12: 387-396Crossref PubMed Scopus (52) Google Scholar), T7 DNA-dependent RNA polymerase (DdRP) utilizes RNA templates (22Arnaud-Barbe N. Cheynet-Sauvion V. Oriol G. Mandrand B. Mallet F. Nucleic Acids Res. 1998; 26: 3550-3554Crossref PubMed Scopus (40) Google Scholar), and poliovirus RdRP utilizes DNA primers (20Arnold J.J. Ghosh S.K. Cameron C.E. J. Biol. Chem. 1999; 274: 37060-37069Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Template preference becomes even more ambiguous when alternative divalent cations, such as Mn2+, are employed (20Arnold J.J. Ghosh S.K. Cameron C.E. J. Biol. Chem. 1999; 274: 37060-37069Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). This “identity crisis” of polymerases regarding template utilization is not too surprising given the existence of enzymes like reverse transcriptases (RTs) that bridge both worlds (23Joyce C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1619-1622Crossref PubMed Scopus (189) Google Scholar). Moreover, the ease of polymerases to move from one template type to another was probably a driving force for the evolution of specific protein-nucleic acid and protein-protein interactions as an obligatory step for the initiation of transcription, replication, and repair (24Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman and Co., New York1991Google Scholar). In contrast to template selection, nucleotide selection is more stringent under physiological conditions. For example, T7 DdRP exhibits an 80-fold preference for rNTPs relative to 2′-dNTPs (25Huang Y. Eckstein F. Padilla R. Sousa R. Biochemistry. 1997; 36: 8231-8242Crossref PubMed Scopus (89) Google Scholar). KF exhibits a 103 to 106-fold preference for 2′-dNTPs (26Minnick D.T. Astatke M. Joyce C.M. Kunkel T.A. J. Biol. Chem. 1996; 271: 24954-24961Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 27Astatke M. Grindley N.D. Joyce C.M. J. Mol. Biol. 1998; 278: 147-165Crossref PubMed Scopus (99) Google Scholar, 28Astatke M. Ng K. Grindley N.D. Joyce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3402-3407Crossref PubMed Scopus (181) Google Scholar). The reverse transcriptases from human immunodeficiency virus (HIV-1) and Moloney murine leukemia virus (MMLV) exhibit a 105-fold preference for 2′-dNTPs (29Zinnen S. Hsieh J.C. Modrich P. J. Biol. Chem. 1994; 269: 24195-24202Abstract Full Text PDF PubMed Google Scholar, 30Gao G. Orlova M. Georgiadis M.M. Hendrickson W.A. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 407-411Crossref PubMed Scopus (162) Google Scholar). The use of Mn2+ as divalent cation permits all classes of polymerase to incorporate one or two nucleotides of the incorrect sugar configuration (31Rienitz A. Grosse F. Blocker H. Frank R. Krauss G. Nucleic Acids Res. 1985; 13: 5685-5695Crossref PubMed Scopus (6) Google Scholar, 32Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4076-4080Crossref PubMed Scopus (256) Google Scholar, 33Beard W.A. Minnick D.T. Wade C.L. Prasad R. Won R.L. Kumar A. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1996; 271: 12213-12220Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 34Astatke M. Grindley N.D.F. Joyce C.M. J. Biol. Chem. 1995; 270: 1945-1954Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 35Spence R.A. Kati W.M. Anderson K.S. Johnson K.A. Science. 1995; 267: 988-993Crossref PubMed Scopus (461) Google Scholar, 36Brandis J.W. Edwards S.G. Johnson K.A. Biochemistry. 1996; 35: 2189-2200Crossref PubMed Scopus (43) Google Scholar). However, processive incorporation of nucleotides of the incorrect sugar configuration is not tolerated (37Lewis D.A. Bebenek K. Beard W.A. Wilson S.H. Kunkel T.A. J. Biol. Chem. 1999; 274: 32924-32930Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar,38Boyer P.L. Sarafianos S.G. Arnold E. Hughes S.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3056-3061Crossref PubMed Scopus (56) Google Scholar). The molecular basis for nucleotide selection by polymerases has been a topic of considerable interest recently (39Kaushik N. Singh K. Alluru I. Modak M.J. Biochemistry. 1999; 38: 2617-2627Crossref PubMed Scopus (18) Google Scholar, 40Bonnin A. Lazaro J.M. Blanco L. Salas M. J. Mol. Biol. 1999; 290: 241-251Crossref PubMed Scopus (74) Google Scholar, 41Harris D. Kaushik N. Pandey P.K. Yadav P.N. Pandey V.N. J. Biol. Chem. 1998; 273: 33624-33634Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 42Kaushik N. Harris D. Rege N. Modak M.J. Yadav P.N. Pandey V.N. Biochemistry. 1997; 36: 14430-14438Crossref PubMed Scopus (27) Google Scholar, 43Gutierrez-Rivas M. Ibanez A. Martinez M.A. Domingo E. Menendez-Arias L. J. Mol. Biol. 1999; 290: 615-625Crossref PubMed Scopus (24) Google Scholar). This interest has resulted from the development of structural models for DNA-dependent DNA polymerases and a DdRP in complex with various substrates (e.g. primer, template, and/or nucleotide). These studies have uncovered interactions between the enzyme and nucleotide that may be important during the selection process (5Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1100) Google Scholar, 6Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (479) Google Scholar, 7Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1355) Google Scholar, 8Doublie S. Ellenberger T. Curr. Opin. Struct. Biol. 1998; 8: 704-712Crossref PubMed Scopus (174) Google Scholar, 9Boyer P.L. Ferris A.L. Clark P. Whitmer J. Frank P. Tantillo C. Arnold E. Hughes S.H. J. Mol. Biol. 1994; 243: 472-483Crossref PubMed Scopus (82) Google Scholar). Construction and characterization of site-directed mutants of KF, HIV-1 RT, and MMLV RT have confirmed the structural predictions by altering the 2′-dNTP/rNTP preference of these enzymes. The 2′-dNTP-utilizing enzymes use a steric gating mechanism to decrease the affinity of the enzyme for rNTPs (27Astatke M. Grindley N.D. Joyce C.M. J. Mol. Biol. 1998; 278: 147-165Crossref PubMed Scopus (99) Google Scholar, 28Astatke M. Ng K. Grindley N.D. Joyce C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3402-3407Crossref PubMed Scopus (181) Google Scholar, 40Bonnin A. Lazaro J.M. Blanco L. Salas M. J. Mol. Biol. 1999; 290: 241-251Crossref PubMed Scopus (74) Google Scholar). The steric gate is formed, in part, by a residue found in structural motif A (motif designations are as defined by Hansen et al. (1Hansen J.L. Long A.M. Schultz S.C. Structure. 1997; 5: 1109-1122Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar)) of the palm subdomain (KF Glu-710, HIV-1 RT Tyr-115, MMLV RT Phe-155). Structural motif B of the palm subdomain may also participate in this process (43Gutierrez-Rivas M. Ibanez A. Martinez M.A. Domingo E. Menendez-Arias L. J. Mol. Biol. 1999; 290: 615-625Crossref PubMed Scopus (24) Google Scholar). The mechanism employed by rNTP-utilizing enzymes is not fully understood. A steric gating mechanism has been proposed for T7 DdRP. Succinctly, it has been suggested that a water molecule bound to Tyr-639, a residue that occludes the nucleotide-binding pocket, is displaced as a consequence of rNTP binding. Displacement of this water molecule results in movement of Tyr-639 out of the pocket, thereby permitting productive rNTP binding. The absence of a 2′-OH would not permit induction of this conformational transition, thereby creating a steric block to productive binding of 2′-dNTPs (25Huang Y. Eckstein F. Padilla R. Sousa R. Biochemistry. 1997; 36: 8231-8242Crossref PubMed Scopus (89) Google Scholar, 44Brieba L.G. Sousa R. Biochemistry. 2000; 39: 919-923Crossref PubMed Scopus (34) Google Scholar). Although this model is based upon steady-state kinetic analysis of T7 RNA polymerase derivatives, a water molecule and movement of Tyr-639 have been observed crystallographically (45Cheetham G.M. Jeruzalmi D. Steitz T.A. Nature. 1999; 399: 80-83Crossref PubMed Scopus (273) Google Scholar, 46Cheetham G.M. Steitz T.A. Science. 1999; 286: 2305-2309Crossref PubMed Scopus (289) Google Scholar). An alternative model has been proposed recently for rNTP selection by T7 DdRP based solely upon structural observations. Selection for rNTP binding appears to be mediated by a hydrogen-bonding network consisting of the 2′-OH and side chains of the enzyme (His-784 and Tyr-639). Such a network is more consistent with the 80-fold preference of this enzyme for rNTPs (25Huang Y. Eckstein F. Padilla R. Sousa R. Biochemistry. 1997; 36: 8231-8242Crossref PubMed Scopus (89) Google Scholar, 47Rechinsky V.O. Kostyuk D.A. Tunitskaya V.L. Kochetkov S.N. FEBS Lett. 1992; 306: 129-132Crossref PubMed Scopus (12) Google Scholar). An 80-fold difference in specificity corresponds to a free energy difference of approximately 3 kcal/mol, a reasonable value for one or two hydrogen bonds (67Fersht A. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Engineering. W. H. Freeman and Co., New York1999: 30Google Scholar). Moreover, as discussed above, steric mechanisms yield specificity differences that are, on average, 4000-fold greater than that observed for this enzyme. Aspects of these two models are mutually exclusive. Analysis of His-784 derivatives under conditions in which 2′-dNTP incorporation by the wild-type enzyme is observed should help to distinguish between these two models (45Cheetham G.M. Jeruzalmi D. Steitz T.A. Nature. 1999; 399: 80-83Crossref PubMed Scopus (273) Google Scholar). Currently, information regarding the mechanism of nucleotide selection by the RdRP is not available. Our previous work has shown that the RdRP from poliovirus utilizes rNTPs at least 121-fold more efficiently than 2′-dNTPs (48Arnold J.J. Cameron C.E. J. Biol. Chem. 2000; 275: 5329-5336Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). This value is similar to that determined for T7 DdRP. In addition, Hansen et al. have predicted the use of a hydrogen-bonding network to select for rNTP binding based upon the unliganded structure of this enzyme (1Hansen J.L. Long A.M. Schultz S.C. Structure. 1997; 5: 1109-1122Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). In this report, we have used the structure for the ternary complex of HIV-1 RT to develop a model for the ternary complex of poliovirus RNA polymerase. In addition, we use biochemical and biological analysis of site-directed mutants of 3Dpol to test predictions of this model. This analysis demonstrates a role for conserved structural motifs A and B in 2′-dNTP/rNTP selection by the RdRP. In addition, we provide additional support for the biological relevance of the primer-template (sym/sub) system developed to study the RdRP from poliovirus in vitro(48Arnold J.J. Cameron C.E. J. Biol. Chem. 2000; 275: 5329-5336Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). [α-32P]UTP (>6,000 Ci/mmol) was from NEN Life Science Products; [γ-32P]ATP (>7,000 Ci/mmol) was from ICN; nucleoside 5′-triphosphates (ultrapure solutions) were from Amersham Pharmacia Biotech, Inc.; all DNA oligonucleotides and T4 DNA ligase were from Life Technologies, Inc.; all RNA oligonucleotides were from Dharmacon Research, Inc. (Boulder, CO); restriction enzymes, T4 polynucleotide kinase, and Deep Vent DNA polymerase were from New England Biolabs, Inc.; polyethyleneimine-cellulose TLC plates were from EM Science; and 2.5-cm DE81 filter paper discs were from Whatman. All other reagents were of the highest grade available from Sigma or Fisher. The coordinates for the HIV-1 RT ternary complex (1rtd) and 3Dpol (1rdr) are available from the Research Collaboratory for Structural Bioinformatics. Superpositioning of the two structures was performed using lsqkab from the CCP4 suite of programs (49Bailey S. Acta Crystallogr. Sec. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar). Structural alignments were initially performed using the thumb and palm subdomains. Final superpositioning of the two structures was confined to structural motifs A (3Dpol residues 233–240), B (), C (324), and E (). The final positions of C-α atoms in the four structural motifs had a root mean square deviation ranging from 0.9 to 1.8 Å. 3Dpol residues were inserted into the structurally analogous positions of HIV-1 RT using the program O (50Jones T.A. Zhou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. D Biol. Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar). Residues having the same identity in both structures were not altered from those observed in the HIV-1 RT structure. Amino acids unique to 3Dpol were manually set in position based on their orientation in the unliganded 3Dpol structure. In some instances, the side chains were adjusted to eliminate steric contact with neighboring residues. 2′-OHs were inserted into both the primer and template strands of the nucleic acid within the polymerase active site as well as the incoming nucleotide. Bond angles for the 2′-OHs were adopted from various RNA structures determined using NMR and x-ray crystallography obtained from the Research Collaboratory for Structural Bioinformatics. Within the vicinity of the active site, DNA in the HIV-1 RT structure adopts an A-form conformation causing the sugar pucker to switch from C2′-endo to C3′-endo; hence, modification of the sugar geometry was not necessary. Nucleotide bases of the RNA were modified to correspond to that of sym/sub (48Arnold J.J. Cameron C.E. J. Biol. Chem. 2000; 275: 5329-5336Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), 5′-GCAUGGGCCC-3′, and the incoming nucleotide was modified to ATP, the first nucleotide incorporated into sym/sub. Two additional regions (comprising residues 163–202) were modeled into the structure based on a partial structural and sequence alignment. Region I, residues 175–202, was identified by superpositioning of the 3Dpol and HIV-1 RT structures and consists of an extended α-helix that runs underneath the 3′-end of the template strand. Region II comprises residues 163–174 (which are absent from the 3Dpol structure), which represent the active site side of the fingers subdomain. Energy minimizations were performed on the entire structure, comprising both modified and unmodified regions, using the program CNS SOLVE (51Brunger A.T. Acta Crystallogr. Sec. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16949) Google Scholar). Initial attempts at energy minimization were performed on the modified region of the structure only; however, upon completion of the first cycle, gross distortions of the molecule were observed. The modified region was reinserted into the entire HIV-1 RT structure, and energy minimizations were repeated. The additional structure eliminated distortions in the molecule, allowing the protein side chains to relax into positions void of unfavorable, steric contact. Iterative cycles of minimization, a total of 10, were performed using the constant temperature algorithm. The final settings for the energy minimization follow. The Cartesian (restrained) molecular dynamics algorithm was utilized at a constant temperature (298 K) using the coupled temperature control method (52Berendsen R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23173) Google Scholar). 10,000 molecular dynamics steps were performed at 0.0005-ps intervals. The dielectric was set to 1 (the default value), and the number of trials utilizing different initial velocities was set to 1. The output files from each cycle were superimposed to observe side chain and nucleic acid motions, which were most apparent for side chains and nucleotides not involved in protein or nucleic acid interactions. Upon completion of the final cycle of minimization, the modified region was removed from the structure, and side chain geometry was checked using the program PROCHECK (53Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Finally, the modified regions of the HIV-1 RT structure, as well as nucleic acid, nucleotide, and Mg2+ ions were removed from the file and used to generate a new Protein Data Bank file (3DRTSS). Mutations were introduced into a modified 3Dpol-coding sequence by using overlap-extension PCR (54Aiyar A. Xiang Y. Leis J. Methods Mol. Biol. 1996; 57: 177-191PubMed Google Scholar) and expressed in Escherichia coli by using a ubiquitin fusion system. The ubiquitin fusion system, PCR conditions, and modified gene have been described previously (55Gohara D.W. Ha C.S. Ghosh S.K.B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expression Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar). The D238F clone was engineered such that it contained a silent NheI site. The sequence of the forward oligonucleotide is 5′-GAC TAC ACA GGG TATTTC GCT AGC CTC AGC CCT-3′; the codon changing Asp to Phe is underlined, and the NheI site is in boldface type. A wild-type reverse oligonucleotide was employed (oligonucleotide 10, Table I). Briefly, two separate PCR reactions were performed: one reaction with the pET-Ub-SacII for oligonucleotide and the Asp-238 WT rev; the other with D238 wild-type rev for and pET-3D-BamHI rev. Both reactions employed pET26b-Ub-3D (55Gohara D.W. Ha C.S. Ghosh S.K.B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expression Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar) as template. Products were purified by agarose gel electrophoresis and used as the template in the next round of PCR with a 1:10 molar ratio of the wild-type:D238F-modified fragments. The AflII for andAvrII reverse oligonucleotides were used as the sole primers for this cycle of PCR. Product was purified, digested withAvrII and AflII, and ligated into pET26b-Ub-3D that had been digested with the same restriction enzymes. Plasmids were screened for the presence of the NheI site. The remaining mutant 3Dpol genes were constructed by using PCR as described above and subcloned into the D238F vector between theAflII and AvrII restriction sites and screened for the loss of the NheI site. Mutations were confirmed by DNA sequencing (Nucleic Acid Facility, Pennsylvania State University).Table IOligonucleotides used in this studyOligonucleotide no.Oligonucleotide nameSequence 1pET-Ub-SacII for5′-GCG GAA TTC CCG CGG TGG AGG TGA AAT CCA GTG G-3′ 2pET-Ub-BamH I rev5′-GCG TCT AGA GGA TCC ACC GCG GAG-3′ 33D AflII for5′-AAA AAC GAT CCC AGG CTT AAG ACA GAC TTT-3′ 43D AvrII rev5′-CCT GAG TGT TCC TAG GAT CTT TAG T-3′ 5D238A for5′-GAC TAC ACA GGG TAT GCT GCA TCT CTC AGC CCT-3′ 6D238E for5′-GAC TAC ACA GGG TAT GAA GCA TCT CTC AGC CCT-3′ 7D238F Nhe for5′-GAC TAC ACA GGG TAT TTC GCA TCT CTC AGC CCT-3′ 8D238N for5′-GAC TAC ACA GGG TAT AAC GCA TCT CTC AGC CCT-3′ 9D238V for5′-GAC TAC ACA GGG TAT GTT GCA TCT CTC AGC CCT-3′10D238 wild-type rev5′-AGG GCT GAG AGA TGC ATC ATA CCC TGT GTA GTC-3′11N297A for5′-GGC ACT TCA ATT TTT GCT TCA ATG ATT AAC AAC-3′12N297D for5′-GGC ACT TCA ATT TTT GAC TCA ATG ATT AAC AAC-3′13N297F for5′-GGC ACT TCA ATT TTT TTC TCA ATG ATT AAC AAC-3′14N297Q for5′-GGC ACT TCA ATT TTT CAG TCA ATG ATT AAC AAC-3′15N297V for5′-GGC ACT TCA ATT TTT GTT TCA ATG ATT AAC AAC-3′16N297 wild-type rev5′-GTT GTT AAT CAT TGA GTT AAA AAT TGA AGT GTT-3′17pUC18BslII top5′-GAT CCA GAT CTA GTA CTG-3′18pUC18BglII bot5′-AAT TCA GTA CTA GAT CTG-3′19pMoEcoRI rev5′-GAA TTA AAT CAT CGA TGA ATT CGG GCC C-3′20pMoBglII for5′-GAA GTG GAG ATC TTG GAT GCC AAA GCG-3′21N-term-Ub5′-ACG CTG TCT GAT TAC AAC-3′22pET-3D-rev5′-TTG GCT TGA CTC ATT TTA GTA AGG ATC CGA ATT CCG C-3′ Open table in a new tab 3Dpol derivatives were expressed and purified as described previously (55Gohara D.W. Ha C.S. Ghosh S.K.B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expression Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar) with the following modifications. 100-ml cultures were lysed by using a French press, nucleic acid was removed by precipitation with polyethyleneimine, and supernatants were clarified by ultracentrifugation (55Gohara D.W. Ha C.S. Ghosh S.K.B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expression Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar). 3Dpol was precipitated by the addition of solid ammonium sulfate to 40% saturation. Recovered pellets were suspended and passed over a 3-ml phosphocellulose column. Bound protein was eluted from the phosphocellulose column by using16 column volume (500 μl) of 50 mm HEPES, pH 7.5, 10 mm dithiothreitol, 20% glycerol, 0.1% Nonidet P-40, and 200 mm NaCl. The proteins were >90% pure based upon SDS-polyacrylamide gel electrophoresis analysis. Two of the derivatives (D238A and N297A) were purified using the complete purification procedure (55Gohara D.W. Ha C.S. Ghosh S.K.B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expression Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar) to >95% purity. The N297F derivative was not soluble when induced in E. coli at 25 °C. The concentration of all 3Dpol derivatives was determined by absorbance at 280 nm using a calculated extinction coefficient of 71,480m−1 cm−1(56Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5035) Google Scholar). The concentration of enzyme stocks prepared by using the abbreviated procedure ranged from 43 to 51 μm. [α-32P]UTP was diluted to 0.1 μCi/μl in distilled deionized H2O, and 1 μl was spotted in triplicate onto TLC plates. TLC plates were developed in 0.3 m potassium phosphate, pH 7.0, dried, and exposed to a PhosphorImager screen (Molecular Dynamics, Inc., Sunnyvale, CA). Imaging and quantitation were performed by using the ImageQuant software from Molecular Dynamics. The purity was used to correct the specific activity of UTP in reactions in order to calculate accurate concentrations of product. Reactions contained 50 mm HEPES, pH 7.5, 10 mm 2-mercaptoethanol, 5 mm MgCl2 or MnCl2, 60 μm ZnCl2, 500 μm UTP, 0.4 μCi/μl [α32-P]UTP, 1.8 μmdT15/2 μm rA30 primer-template, and 3Dpol. Reactions were carried out in a total volume of 25 μl with 250 ng of enzyme at 30 °C for 5 min. Reactions were quenched by the addition of 5 μl of 0.5 m EDTA. 10 μl of the quenched reaction was spotted onto DE81 filter paper discs and dried completely. The discs were washed three times for 10 min in 250 ml of 5% dibasic sodium phosphate and rinsed in absolute ethanol. Bound radioactivity was quantitated by liquid scintillation counting in 5 ml of EcoScint scintillation fluid (National Diagnostics). RNA oligonucleotides were end-labeled using [γ-32P]ATP and T4 polynucleotide kinase essentially as specified by the manufacturer. Reactions typically contained 11 μm[γ-32P]ATP, 10 μm RNA oligonucleotide, and 0.4 units/μl T4 polynucleotide kinase. Unincorporated nucleotide was removed by passing the" @default.
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• W2055140930 date "2000-08-01" @default.
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• W2055140930 title "Poliovirus RNA-dependent RNA Polymerase (3Dpol)" @default.
• W2055140930 cites W1493170168 @default.
• W2055140930 cites W1507189859 @default.
• W2055140930 cites W1520647240 @default.
• W2055140930 cites W1545429574 @default.
• W2055140930 cites W1605800306 @default.
• W2055140930 cites W1907331499 @default.
• W2055140930 cites W1932632320 @default.
• W2055140930 cites W1939421214 @default.
• W2055140930 cites W1967021038 @default.
• W2055140930 cites W1967798978 @default.
• W2055140930 cites W1971364137 @default.
• W2055140930 cites W1973161154 @default.
• W2055140930 cites W1973711348 @default.
• W2055140930 cites W1975705587 @default.
• W2055140930 cites W1977599839 @default.
• W2055140930 cites W1978114375 @default.
• W2055140930 cites W1979155909 @default.
• W2055140930 cites W1981218571 @default.
• W2055140930 cites W1986191025 @default.
• W2055140930 cites W1990083018 @default.
• W2055140930 cites W1995017064 @default.
• W2055140930 cites W2003469154 @default.
• W2055140930 cites W2005986251 @default.
• W2055140930 cites W2009865515 @default.
• W2055140930 cites W2012419248 @default.
• W2055140930 cites W2013083986 @default.
• W2055140930 cites W2018542294 @default.
• W2055140930 cites W2018872766 @default.
• W2055140930 cites W2025010288 @default.
• W2055140930 cites W2025055438 @default.
• W2055140930 cites W2026491313 @default.
• W2055140930 cites W2028442352 @default.
• W2055140930 cites W2030009404 @default.
• W2055140930 cites W2030349151 @default.
• W2055140930 cites W2031536871 @default.
• W2055140930 cites W2035266068 @default.
• W2055140930 cites W2041421251 @default.
• W2055140930 cites W2044154059 @default.
• W2055140930 cites W2045378360 @default.
• W2055140930 cites W2050076455 @default.
• W2055140930 cites W2056307424 @default.
• W2055140930 cites W2057974881 @default.
• W2055140930 cites W2066076729 @default.
• W2055140930 cites W2067371216 @default.
• W2055140930 cites W2069583223 @default.
• W2055140930 cites W2084702672 @default.
• W2055140930 cites W2087814255 @default.
• W2055140930 cites W2089142368 @default.
• W2055140930 cites W2089891949 @default.
• W2055140930 cites W2089898350 @default.
• W2055140930 cites W2092348568 @default.
• W2055140930 cites W2098419010 @default.
• W2055140930 cites W2106870343 @default.
• W2055140930 cites W2118848130 @default.
• W2055140930 cites W2119499189 @default.
• W2055140930 cites W2121255902 @default.
• W2055140930 cites W2128783473 @default.
• W2055140930 cites W2131505205 @default.
• W2055140930 cites W2134249956 @default.
• W2055140930 cites W2138892620 @default.
• W2055140930 cites W2149649955 @default.
• W2055140930 cites W2210677056 @default.
• W2055140930 cites W2580627695 @default.
• W2055140930 cites W282873500 @default.
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