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• W2080467264 abstract "MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif (249GTTRD253), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly249 and Thr251. On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression. MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif (249GTTRD253), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly249 and Thr251. On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression. GTPases are involved in many different and essential cellular processes, including protein synthesis and translocation, membrane trafficking, signal transduction, and cell cycle control (1Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2655) Google Scholar, 2Leipe D.D. Wolf Y.I. Koonin E.V. Aravind L. J. Mol. Biol. 2002; 317: 41-72Crossref PubMed Scopus (834) Google Scholar). The common property shared by these proteins is the presence of a structural module, the G-domain, which is involved in the switching of the protein between a GTP-bound and a GDP-bound conformation. This conformational switch is crucial for the function of all GTPases (3Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1335) Google Scholar). Three-dimensional structures of members from the G protein superfamily indicate that the conserved residues of the G domain (grouped in motifs G1 to G4, Fig. 1) are invariably involved in binding of guanine nucleotides and Mg2+, hydrolysis of GTP, or controlling the conformational change. This change is primarily confined to two highly flexible regions called switches I and II, which include the G2 and G3 motifs, respectively. The GTPase cycle is regulated by the intrinsic properties of each GTPase, as well as by the specific factors that the GTPase interacts with. Thus, GAPs 1The abbreviations used are: GAP, GTPase-activating protein; Pi, inorganic phosphate; cmnm, carboximethylaminomethyl; mnm5U34, 5-methylaminomethyl uridine at position 34; mnm5s2U, 5-methylaminomethyl-2-thiouridine; mnm5s2U34, 5-methylaminomethyl-2-thiouridine at position 34; cmnm5s2U34, 5-carboximethylaminomethyl-2-thiouridine at position 34; s2U, 2-thiouridine; s2U34, 2-thiouridine at position 34; mant, methylanthraniloyl; GTPγS, guanosine 5′-3-O-(thio)triphosphate. 1The abbreviations used are: GAP, GTPase-activating protein; Pi, inorganic phosphate; cmnm, carboximethylaminomethyl; mnm5U34, 5-methylaminomethyl uridine at position 34; mnm5s2U, 5-methylaminomethyl-2-thiouridine; mnm5s2U34, 5-methylaminomethyl-2-thiouridine at position 34; cmnm5s2U34, 5-carboximethylaminomethyl-2-thiouridine at position 34; s2U, 2-thiouridine; s2U34, 2-thiouridine at position 34; mant, methylanthraniloyl; GTPγS, guanosine 5′-3-O-(thio)triphosphate. stimulate GTPase reaction by several orders of magnitude and guanine nucleotide exchange factors and guanine nucleotide-dissociation inhibitors regulate the nucleotide exchange (3Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1335) Google Scholar). Regulatory GTPases, like Ras-related proteins, are in an active state when GTP-bound; the binding of GTP causes a conformational change in the proteins that allows interaction with a target molecule or effector; upon GTP hydrolysis, they become inactive. The GTPase cycle of Ras proteins requires participation of GAPs and guanine nucleotide exchange factors, because these GTPases typically show a very low intrinsic hydrolase activity and a very high affinity for guanine nucleotides. In most members of Ras family, GAPs stabilize the transition state of the reaction by supplying a so-called arginine finger (which stabilizes the negative charge development at the phosphate groups of GTP during the hydrolysis reaction) and enhancing stability of amino acids located in switches I and II (3Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1335) Google Scholar, 4Bourne H.R. Nature. 1997; 389: 673-674Crossref PubMed Scopus (67) Google Scholar, 5Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmüller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1163) Google Scholar, 6Geyer M. Wittinghofer A. Curr. Opin. Struct. Biol. 1997; 7: 786-792Crossref PubMed Scopus (140) Google Scholar, 7Li G. Zhang X.C. J. Mol. Biol. 2004; 340: 921-932Crossref PubMed Scopus (68) Google Scholar). A glutamine adjacent to motif G3 is also crucial to stabilize the transition state and orient the attacking water molecule, being assisted in this respect by the arginine finger. Gα subunits of trimeric G proteins use a similar mechanism for GTP hydrolysis, but the catalytic invariant arginine is provided in cis from a helical domain of the GTPase polypeptide (3Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1335) Google Scholar). Other cases of GTPases carrying putative catalytic arginines in “cis” have been recently reported (8Zhang B. Zhang Y. Collins C.C. Johnson D.I. Zheng Y. J. Biol. Chem. 1999; 274: 2609-2612Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 9Mohr D. Wintermeyer W. Rodnina M. EMBO J. 2000; 19: 3458-3464Crossref PubMed Scopus (30) Google Scholar). Interestingly, there are several data pointing out that multiple GTP hydrolysis mechanisms independent of Gln and Arg are likely to exist (3Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1335) Google Scholar, 10Daumke O. Weyand M. Chakrabarti P.P. Vetter I.R. Wittinghofer A. Nature. 2004; 429: 197-201Crossref PubMed Scopus (114) Google Scholar, 11Seewald M.J. Körner C. Wittinghofer A. Vetter I.R. Nature. 2002; 415: 662-666Crossref PubMed Scopus (164) Google Scholar). The evolutionarily conserved MnmE protein is a GTPase with a tRNA modification function in Escherichia coli (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar, 13Yim L. Martínez-Vicente M. Villarroya M. Aguado C. Knecht E. Armengod M.E. J. Biol. Chem. 2003; 278: 28378-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). MnmE is involved in the modification of the wobble-position uridine (U34) of tRNAs that read codons ending with A or G, in the mixed codon family boxes, i.e. tRNAmnm5s2UUULys, tRNAmnm5s2UUCGlu, tRNAcmnm5s2UUGGln, tRNAcmnm5UmAALeu, and tRNAmnm5UCUArg. In the modification pathway of these tRNAs, MnmE controls, together with GidA, the addition of the cmnm group in position 5 of the U34, although the precise role of both proteins in the modification reaction is still unknown (Fig. 2). In tRNAmnm5s2UUULys and tRNAmnm5s2UUCGlu, MnmC transforms the cmnm5 group into the final mnm5 modification, meanwhile MnmA, together with IscS, carries out thiolation in the 2-position of the wobble uridine (14Hagervall T.G. Edmonds C.G. McCloskey J.A. Björk G.R. J. Biol. Chem. 1987; 262: 8488-8495Abstract Full Text PDF PubMed Google Scholar, 15Kambampati R. Lauhon C.T. Biochemistry. 2003; 42: 1109-1117Crossref PubMed Scopus (130) Google Scholar, 16Bujnicki J.M. Oudjama Y. Roovers M. Owczarek S. Caillet J. Droogmans L. RNA (N. Y.). 2004; 10: 1236-1242Crossref PubMed Scopus (36) Google Scholar). Modifications in the 2- and 5-positions occur independently of each other; thus, thiolation may precede or follow the synthesis of the side chain at position 5. Note that non-thiolated derivatives are present in tRNAmnm5UmAALeu and tRNAmnm5UCUArg, whereas tRNAcmnm5s2UUGGln contains cmnm5s2. 2G. R. Björk, unpublished observation. 2G. R. Björk, unpublished observation. When selenium is available in the growth medium, sulfur at position 2 may be replaced by selenium in a reaction dependent on SelD and YbbB (17Ching W.-M. Tsai L. Wittwer A.J. Curr. Top. Cell Regul. 1985; 27: 497-507Crossref PubMed Scopus (14) Google Scholar, 18Wittwer A.J. Stadtman T.C. Arch. Biochem. Biophys. 1986; 248: 540-550Crossref PubMed Scopus (40) Google Scholar, 19Wolfe M.D. Ahmed F. Lacourciere G.M. Lauhon C.T. Stadtman T.C. Larson T.J. J. Biol. Chem. 2004; 279: 1801-1809Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). It has been shown that modifications at position 2, but not at position 5, are important for aminoacylation of tRNAs (20Ching W.-M. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 374-377Crossref PubMed Scopus (32) Google Scholar, 21Elseviers D. Petrullo L.A. Gallagher P. Nucleic Acids Res. 1984; 12: 3521-3534Crossref PubMed Scopus (100) Google Scholar, 22Krüger M.K. Sørensen M.A. J. Mol. Biol. 1998; 284: 609-620Crossref PubMed Scopus (54) Google Scholar), whereas modification in both 2- and 5-positions function in the codon recognition process (17Ching W.-M. Tsai L. Wittwer A.J. Curr. Top. Cell Regul. 1985; 27: 497-507Crossref PubMed Scopus (14) Google Scholar, 18Wittwer A.J. Stadtman T.C. Arch. Biochem. Biophys. 1986; 248: 540-550Crossref PubMed Scopus (40) Google Scholar, 22Krüger M.K. Sørensen M.A. J. Mol. Biol. 1998; 284: 609-620Crossref PubMed Scopus (54) Google Scholar, 23Kramer G.F. Ames B.N. J. Bacteriol. 1988; 170: 736-743Crossref PubMed Google Scholar, 24Brégeon D. Colot V. Radman M. Taddei F. Genes Dev. 2001; 15: 2295-2306Crossref PubMed Scopus (126) Google Scholar, 25Curran J.M. Grosjean H. Benne R. Modification and Editing of RNA. American Society for Microbiology, Washington, D. C.1998: 493-516Google Scholar, 26Urbonavicius J. Qian Q. Durand J.M.B. Hagervall T.G. Björk G.R. EMBO J. 2001; 20: 4863-4873Crossref PubMed Scopus (360) Google Scholar, 27Yarian C. Townsend H. Czestkowski W. Sochacka E. Malkiewicz A.J. Guenther R. Miskiewicz A. Agris P.F. J. Biol. Chem. 2002; 277: 16391-16395Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Unlike the G domain-only proteins of the Ras family, MnmE is a medium-size protein of 50 kDa that consists of three regions, an ∼220-amino acid N-terminal region, required for self-assembly, a middle GTPase domain of about 160 residues, and an ∼75-amino acid C-terminal region, which contains the only Cys residue present in the protein (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar) (see Fig. 1). Moreover, MnmE can be distinguished from other GTPases like Ras proteins and translation factors by its lower affinity for GTP and GDP, and higher intrinsic rate of GTP hydrolysis (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar, 13Yim L. Martínez-Vicente M. Villarroya M. Aguado C. Knecht E. Armengod M.E. J. Biol. Chem. 2003; 278: 28378-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 28Scrima A. Vetter I.R. Armengod M.E. Wittinghofer A. EMBO J. 2005; 24: 23-33Crossref PubMed Scopus (66) Google Scholar); this peculiar biochemical properties determine that MnmE can perform multiple rounds of GTP hydrolysis in vitro without participation of guanine nucleotide exchange factors or GAPs, but the protein requires very high substrate concentrations for high rates of GTP hydrolysis (Km > 400 μm). Strikingly, the isolated GTPase domain of MnmE roughly conserves the guanine nucleotide binding and GTPase activities of the intact MnmE molecule, which indicates that the MnmE G-domain contains the catalytic residues that explain for the relatively high intrinsic GTPase activity exhibited by the entire protein. A recent mutational analysis indicated that the G domain of MnmE is essential for its tRNA-modifying function, which requires effective hydrolysis of GTP, and not simply GTP binding (13Yim L. Martínez-Vicente M. Villarroya M. Aguado C. Knecht E. Armengod M.E. J. Biol. Chem. 2003; 278: 28378-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Thus, it was suggested that MnmE uses GTP hydrolysis to promote structural rearrangements that convert the protein to its active state. The trigger for the conformational change of GTPases is thought to be universal. In the GTP-bound form, there are two hydrogen bonds from γ-phosphate oxygens to the main-chain NH groups of invariant residues Thr (G2 motif) and Gly (G3 motif). After GTP hydrolysis (which may be or not dependent on an arginine finger), there is a structural rearrangement into a new conformation, mediated by the loss of the interactions of these residues (3Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1335) Google Scholar). The main purpose of this work was to study the role of the G2 motif in the functional mechanism of MnmE as well as to get insights into the peculiar biochemical properties of this protein by mutating the conserved arginines of its G-domain. Bacterial Strains, Phages, Plasmids, and DNA Manipulations— Escherichia coli strains and plasmids used in this study are listed in Table I, unless specified otherwise. Genetic techniques for the construction of strains were performed as described previously (30Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992Google Scholar). λIC718 is a λRZ5 derivative carrying the wild-type mnmE allele under control of promoter Ptac (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar). For DNA manipulations, standard procedures were followed. Derivatives from pIC684 and pIC914 plasmids were obtained by site-directed oligonucleotide mutagenesis with appropriated PCR primers. All constructs were verified by DNA sequencing.Table IE. coli strains and plasmids used in this studyStrain or plasmidDescriptionaThe numbering system used to define E. coli chromosomal fragments present in some plasmids is that employed by Cabedo et al. (12); in such a system, mnmE extends from position 3102 to 4466. Mutant MnmE protein and/or phenotype is indicated between brackets.Origin and/or referenceE. coli strainsDEV16F- thi-1 rel-1 spoT1 lacZ105UAG mnmE192UAG [MnmE Q192X, ValR]21Elseviers D. Petrullo L.A. Gallagher P. Nucleic Acids Res. 1984; 12: 3521-3534Crossref PubMed Scopus (100) Google ScholarJC7623recB21 recC22 sbcB15 sbcC201 Sal-29Bachmann B. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 2460-2488Google ScholarV5701bgl (Sal+)12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar, 29Bachmann B. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 2460-2488Google ScholarMG1655F—D. TouatiIC3647JC7623 (λIC718)See JC7623 (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar)IC4126V5701/pIC755See V5701 (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar)IC4130IC4126 mnmE::kan [MnmE-]See IC4126 (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar)IC4760IC3647 mnmE+, tnaA::kan13Yim L. Martínez-Vicente M. Villarroya M. Aguado C. Knecht E. Armengod M.E. J. Biol. Chem. 2003; 278: 28378-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google ScholarIC4836IC3647 mnmE23, tnaA::kan [MnmE R252A, KanR]This workIC4837IC3647 mnmE102, tnaA::kan [MnmE R275A, KanR]This workIC4903IC3647 mnmE21, tnaA::kan [MnmE T250A, KanR]This workIC4904IC3647 mnmE22, tnaA::kan [MnmE T251A, KanR]This workIC4905IC3647 mnmE20, tnaA::kan [MnmE G249A, KanR]This workIC4955IC3647 mnmE24, tnaA::kan [MnmE D253A, KanR]This workIC5057IC3647 mnmE25, tnaA::kan [MnmE T250S, KanR]This workIC5082IC3647 mnmE26, tnaA::kan [MnmE T251S, KanR]This workIC5083IC3647 mnmE103, tnaA::kan [MnmE R288A, KanR]This workIC5109IC3647 mnmE27, tnaA::kan [MnmE T250S/T251S, KanR]This workIC5110IC3647 mnmE101, tnaA::kan [MnmE R256A, KanR]This workIC5118IC3647 mnmE100, tnaA::kan [MnmE R224A, KanR]This workIC5168IC3647 mnmE28, tnaA::kan [MnmE R252K, KanR]This workIC5357MG1655 tnaA::kan [MnmE+, KanR]This workIC5358MG1655 mnmE::kan [MnmE-, KanR]This workIC5360MG1655 mnmE103, tnaA::kan [MnmE R288A, KanR]This workIC5361MG1655 mnmE25, tnaA::kan [MnmE T250S, KanR]This workIC5362MG1655 mnmE26, tnaA::kan [MnmE T251S, KanR]This workPlasmidspFM1Chromosomal DNA fragment 980-9847 (containing mnmE and tnaA) inserted between the HindIII and EcoRI sites on pT7-12 [MnmE+]12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google ScholarpIC684GST fusion of DNA fragment from 3033 to 5652 (GST-MnmE+ fusion)12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google ScholarpIC755HindIII fragment 980-7374 (containing mnmE) inserted into HindIII-cut pMAK700 (MnmE+, CmR, replicon Ts)12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google ScholarpIC9141.3-kb SalI fragment (KanR determinant) from pUC4K inserted into tnaA (NruI site, nt 5652) on pFM113Yim L. Martínez-Vicente M. Villarroya M. Aguado C. Knecht E. Armengod M.E. J. Biol. Chem. 2003; 278: 28378-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google ScholarpIC935pIC684 derivative containing mnmE10 [MnmE G228A]13Yim L. Martínez-Vicente M. Villarroya M. Aguado C. Knecht E. Armengod M.E. J. Biol. Chem. 2003; 278: 28378-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google ScholarpIC938pIC684 derivative containing mnmE102 [MnmE R275A]This workpIC939pIC684 derivative containing mnmE23 [MnmE R252A]This workpIC1002pIC684 derivative containing mnmE21 [MnmE T250A]This workpIC1003pIC684 derivative containing mnmE22 [MnmE T251A]This workpIC1013pIC684 derivative containing mnmE24 [MnmE D253A]This workpIC1048pIC684 derivative containing mnmE26 [MnmE T251S]This workpIC1050pIC684 derivative containing mnmE103 [MnmE R288A]This workpIC1059pIC684 derivative containing mnmE25 [MnmE T250S]This workpIC1068pIC684 derivative containing mnmE100 [MnmE R224A]This workpIC1070pIC684 derivative containing mnmE27 [MnmE T250S/T251S]This workpIC1072pIC684 derivative containing mnmE101 [MnmE R256A]This workpIC1081pIC684 derivative containing mnmE28 [MnmE R252K]This workpIC979pIC914 derivative containing mnmE23 [MnmE R252A]This workpIC980pIC914 derivative containing mnmE102 [MnmE R275A]This workpIC1012pIC914 derivative containing mnmE20 [MnmE G249A]This workpIC1014pIC914 derivative containing mnmE24 [MnmE D253A]This workpIC1015pIC914 derivative containing mnmE21 [MnmE T250A]This workpIC1016pIC914 derivative containing mnmE22 [MnmE T251A]This workpIC1049pIC914 derivative containing mnmE26 [MnmE T251S]This workpIC1051pIC914 derivative containing mnmE103 [MnmE R288A]This workpIC1060pIC914 derivative containing mnmE25 [MnmE T250S]This workpIC1069pIC914 derivative containing mnmE100 [MnmE R224A]This workpIC1073pIC914 derivative containing mnmE101 [MnmE R256A]This workpIC1082pIC914 derivative containing mnmE28 [MnmE R252K]This worka The numbering system used to define E. coli chromosomal fragments present in some plasmids is that employed by Cabedo et al. (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar); in such a system, mnmE extends from position 3102 to 4466. Mutant MnmE protein and/or phenotype is indicated between brackets. Open table in a new tab Media, Growth Conditions, and Enzyme Assays—Luria-Bertani broth containing 40 μg/ml thymine) and LAT (Luria-Bertani broth containing 20 g of Difco agar per liter) were used for routine cultures and plating of E. coli. Antibiotics were added as recommended (30Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992Google Scholar). IC4126 and derivatives were grown at 30 °C and plated at 30 °C or at 42 °C for viability tests. β-Galactosidase assay was performed as previously described (30Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992Google Scholar). Protein Techniques—Overproduction and purification of glutathione S-transferase fusion proteins was done in DEV16 transformed with plasmid pIC684 or derivatives. Cleavage with thrombin of the chimeras, which were purified by affinity chromatography, allowed the isolation of recombinant wild-type (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar) and mutant MnmE proteins. These proteins were used for the in vitro assays. In this respect, it should be pointed out that native and recombinant forms of the wild-type MnmE protein exhibit identical GTPase properties. 3M. Martínez-Vicente, L. Yim, M. Villarroya, M. Mellado, E. Pérez-Payá, G. R. Björk, and M.-E. Armengod, unpublished data. SDS-PAGE and immunoblotting were carried out essentially as described (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar). Before use, the anti-MnmE antiserum (12Cabedo H. Macián F. Villarroya M. Escudero J.C. Martínez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar) was affinity-purified with nitrocellulose-bound MnmE. GTPase and Nucleotide-binding Assays—GTP hydrolysis was measured by a colorimetric assay for determination of Pi release, as previously described (13Yim L. Martínez-Vicente M. Villarroya M. Aguado C. Knecht E. Armengod M.E. J. Biol. Chem. 2003; 278: 28378-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). To determine values for Vmax and Km, the data were fitted to the Michaelis-Menten equation using non-linear regression (GraphPad Prism version 3.00 for Windows, GraphPad Software, Inc.). MnmE was titrated against fluorescent mant nucleotides (Jena Bioscience) until saturation was reached. The mant nucleotides (2 μm) were excited at 360 nm, and the fluorescence was monitored at 440 nm (LS 50 B spectrophotometer, PerkinElmer Life Sciences). Unless otherwise indicated, all binding assays were performed at 25 °C in GTPase buffer (50 mm Tris-HCl, pH 7.5, 50 mm KCl, 2 mm MgCl2, 5% glycerol). The binding constants (Kd values) were calculated by fitting the curves with non-linear regression, using GraphPad Prism software. Readthrough Measurements—Misreading of the UAG stop codon carried by the lacZ105 gene was monitored by using the β-galactosidase assay as described (13Yim L. Martínez-Vicente M. Villarroya M. Aguado C. Knecht E. Armengod M.E. J. Biol. Chem. 2003; 278: 28378-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Special precautions for very low β-galactosidase activities were taken (21Elseviers D. Petrullo L.A. Gallagher P. Nucleic Acids Res. 1984; 12: 3521-3534Crossref PubMed Scopus (100) Google Scholar, 24Brégeon D. Colot V. Radman M. Taddei F. Genes Dev. 2001; 15: 2295-2306Crossref PubMed Scopus (126) Google Scholar). Analysis of tRNAGlu Modification by Northern Blot—Total tRNA from strains grown to an A600 ∼ 0.6–0.8 was extracted and deacylated (22Krüger M.K. Sørensen M.A. J. Mol. Biol. 1998; 284: 609-620Crossref PubMed Scopus (54) Google Scholar). The tRNA was quantified by absorbance measurement at 260 nm with a Unicam UV-visible spectrophotometer (Helios-β). Northern blots were performed as described (13Yim L. Martínez-Vicente M. Villarroya M. Aguado C. Knecht E. Armengod M.E. J. Biol. Chem. 2003; 278: 28378-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Analysis of Modified Nucleosides in tRNA—Bacterial strains were grown in Luria-Bertani broth at 37 °C to about 5 × 108 cells/ml (A600 ∼ 0.8). The cells were lysed, and total RNA was prepared (31Emilsson V. Kurland C.G. EMBO J. 1990; 9: 4359-4366Crossref PubMed Scopus (92) Google Scholar), dissolved in R200 buffer (100 mm Tris-H3PO4, pH 6.3, 15% ethanol, 200 mm KCl), and applied to a Nucleobond column equilibrated with the same buffer. tRNA was eluted with the same buffer, except that the KCl concentration was raised to 650 mm. The tRNA was precipitated with 2.5 volumes of cold ethanol containing 1% of potassium acetate, washed twice with 70% ethanol, and dried. It was then dissolved in water, and a 75-μg sample was degraded to nucleosides with nuclease P1 (US Biological) followed by treatment with bacterial alkaline phosphatase (32Gehrke C.W. Kuo K.C. J. Chromatogr. 1989; 471: 3-36Crossref PubMed Scopus (200) Google Scholar). The resulting hydrolysate was analyzed by high performance liquid chromatography (32Gehrke C.W. Kuo K.C. J. Chromatogr. 1989; 471: 3-36Crossref PubMed Scopus (200) Google Scholar) on a Develosil C30 column (Phenomenex). The chromatograms were scanned at specific wavelengths to optimize the detection of thiolated nucleosides. It should be pointed out that Luria-Bertani broth is deficient in selenium (18Wittwer A.J. Stadtman T.C. Arch. Biochem. Biophys. 1986; 248: 540-550Crossref PubMed Scopus (40) Google Scholar); therefore, MnmE-specific tRNAs isolated from strains grown in this medium mostly carry sulfur at position 2. Mutational Analysis of the MnmE Switch I Region—In the switch I region of GTP-binding proteins, an invariant Thr is the only residue of the G2 motif conserved among GTPase families (Fig. 1). It is usually involved, via its side-chain hydroxyl in the coordination of the crucial Mg2+ and, via its main chain NH, in contacting the γ-phosphate. Other residues of the G2 motif are highly conserved within each GTPase family, but not between different families. Alignment of members of the MnmE family indicates that motif GTTRD, between G1 and G3, is absolutely conserved and, therefore, it appears as the putative G2 signature sequence of this family (Fig. 1). To analyze its role in the biochemical properties of MnmE, we firstly changed each residue in this motif to alanine. To determine whether the mutant proteins were capable of binding guanine nucleotides, we carried out binding studies in solution with fluorescent nucleotides. mant-guanine nucleotides have been shown to be useful probes of G-protein activation and conformational state (33Lin B. Covalle K.L. Maddock J.R. J. Bacteriol. 1999; 181: 5825-5832Crossref PubMed Google Scholar, 34Lin B. Skidmore J.M. Bhatt A. Pfeffer S.M. Pawloski L. Maddock J.R. Mol. Microbiol. 2001;" @default.
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• W2080467264 cites W120175838 @default.
• W2080467264 cites W1482562661 @default.
• W2080467264 cites W1485008208 @default.
• W2080467264 cites W1607300871 @default.
• W2080467264 cites W1672842817 @default.
• W2080467264 cites W1827226170 @default.
• W2080467264 cites W1837690179 @default.
• W2080467264 cites W1963570235 @default.
• W2080467264 cites W1965517840 @default.
• W2080467264 cites W1967483524 @default.
• W2080467264 cites W1970052480 @default.
• W2080467264 cites W1972333514 @default.
• W2080467264 cites W1974607992 @default.
• W2080467264 cites W1977598234 @default.
• W2080467264 cites W1978611087 @default.
• W2080467264 cites W1981222345 @default.
• W2080467264 cites W1981899562 @default.
• W2080467264 cites W1986763488 @default.
• W2080467264 cites W1989233227 @default.
• W2080467264 cites W1992864103 @default.
• W2080467264 cites W1997016195 @default.
• W2080467264 cites W1999249940 @default.
• W2080467264 cites W2000236507 @default.
• W2080467264 cites W2001504395 @default.
• W2080467264 cites W2007242362 @default.
• W2080467264 cites W2014713866 @default.
• W2080467264 cites W2016906651 @default.
• W2080467264 cites W2020346411 @default.
• W2080467264 cites W2022046854 @default.
• W2080467264 cites W2022895983 @default.
• W2080467264 cites W2024684906 @default.
• W2080467264 cites W2030788867 @default.
• W2080467264 cites W2031178514 @default.
• W2080467264 cites W2034304629 @default.
• W2080467264 cites W2040118814 @default.
• W2080467264 cites W2047384586 @default.
• W2080467264 cites W2047627595 @default.
• W2080467264 cites W2051221651 @default.
• W2080467264 cites W2052738911 @default.
• W2080467264 cites W2056838179 @default.
• W2080467264 cites W2059121036 @default.
• W2080467264 cites W2065735398 @default.
• W2080467264 cites W2066598824 @default.
• W2080467264 cites W2068666155 @default.
• W2080467264 cites W2069885328 @default.
• W2080467264 cites W2077167882 @default.
• W2080467264 cites W2079390173 @default.
• W2080467264 cites W2079511743 @default.
• W2080467264 cites W2080583390 @default.
• W2080467264 cites W2082570884 @default.
• W2080467264 cites W2088112980 @default.
• W2080467264 cites W2091803996 @default.
• W2080467264 cites W2092437075 @default.
• W2080467264 cites W2095642827 @default.
• W2080467264 cites W2099828888 @default.
• W2080467264 cites W2104480986 @default.
• W2080467264 cites W2125728906 @default.
• W2080467264 cites W2139723407 @default.
• W2080467264 cites W2142677752 @default.
• W2080467264 cites W2145207673 @default.
• W2080467264 cites W2156571424 @default.
• W2080467264 cites W2160756301 @default.
• W2080467264 cites W2166606571 @default.
• W2080467264 cites W2167144660 @default.
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