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• W2156294872 abstract "NMR was used to study the solution structure of bovine tRNATrp hyperexpressed in Escherichia coli. With the use of 15N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U71A2 by G2C71, U27A43 by G27C43, and G12C23 by U12A23, the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A73and the G12C23 base pair on the D stem, two identity elements on bovine tRNATrp that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G73 and U12A23, respectively, NMR analysis revealed an important conformational change in the U12A23mutant but not in the G73 mutant molecule. Thus A73 appears to be directly recognized by tryptophanyl-tRNA synthetase, and G12C23 represents an important structural determinant. Mg2+ effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg2+ binding sites in tRNATrp. Strong Mg2+ binding modes were associated with the residues G7, s4U8 (where s4U is 4-thiouridine), G12, and U52. The observations that G42 was associated with strong Mg2+ binding in only the U12A23mutant tRNATrp but not the wild type or G73mutant tRNATrp and that the G7, s4U8, G24, and G22imino protons are associated with a two-site Mg2+ binding mode in wild type and G73 mutant but only a one-site mode in the U12A23 mutant established the occurrence of conformational change in the U12A23 mutant tRNATrp. These observations also established the dependence of Mg2+ binding on tRNA conformation and the usefulness of Mg2+ binding sites as conformational probes. The thermal titration of tRNATrp in the presence and absence of 10 mm Mg2+ indicated that overall tRNATrp structure stability was increased by more than 15 °C by the presence of Mg2+. NMR was used to study the solution structure of bovine tRNATrp hyperexpressed in Escherichia coli. With the use of 15N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U71A2 by G2C71, U27A43 by G27C43, and G12C23 by U12A23, the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A73and the G12C23 base pair on the D stem, two identity elements on bovine tRNATrp that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G73 and U12A23, respectively, NMR analysis revealed an important conformational change in the U12A23mutant but not in the G73 mutant molecule. Thus A73 appears to be directly recognized by tryptophanyl-tRNA synthetase, and G12C23 represents an important structural determinant. Mg2+ effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg2+ binding sites in tRNATrp. Strong Mg2+ binding modes were associated with the residues G7, s4U8 (where s4U is 4-thiouridine), G12, and U52. The observations that G42 was associated with strong Mg2+ binding in only the U12A23mutant tRNATrp but not the wild type or G73mutant tRNATrp and that the G7, s4U8, G24, and G22imino protons are associated with a two-site Mg2+ binding mode in wild type and G73 mutant but only a one-site mode in the U12A23 mutant established the occurrence of conformational change in the U12A23 mutant tRNATrp. These observations also established the dependence of Mg2+ binding on tRNA conformation and the usefulness of Mg2+ binding sites as conformational probes. The thermal titration of tRNATrp in the presence and absence of 10 mm Mg2+ indicated that overall tRNATrp structure stability was increased by more than 15 °C by the presence of Mg2+. Although the three-dimensional structures of a number of free and enzyme-bound tRNA molecules have been elucidated with x-ray crystallography (1Robertus J.D. Ladner J.E. Finch J.T. Rhodes D. Brown R.S. Clark B.F.C. Klug A. Nature. 1974; 250: 546-551Crossref PubMed Scopus (793) Google Scholar, 2Kim S. Suddath F. Quigley G. McPherson A. Sussman J. Wang A. Seeman N. Rich A. Science. 1974; 185: 435-440Crossref PubMed Scopus (741) Google Scholar, 3Moras D. Comarmond M.B. Fischer J. Weiss R. Thierry J.C. Ebel J.P. Giegé R. Nature. 1980; 288: 669-674Crossref PubMed Scopus (241) Google Scholar, 4Ruff M. Krishnaswamy S. Boeglin M. Poterszman A. Mitschler A. Podjarny A. Rees B. Thierry J.C. Moras D. Science. 1991; 252: 1682-1689Crossref PubMed Scopus (590) Google Scholar, 5Biou V. Yaremchuk A. Tukalo M. Cusack S. Science. 1994; 263: 1404-1410Crossref PubMed Scopus (420) Google Scholar, 6Rould M.A. Perona J.J. Söll D. Steitz T.A. Science. 1989; 246: 1135-1142Crossref PubMed Scopus (799) Google Scholar), the solution structures of most tRNAs remain undetermined despite their pivotal importance in protein synthesis where they must be recognized with high fidelity by cognate aminoacyl-tRNA synthetases (7Wallis N.G. Dardel F. Blanquet S. Biochemistry. 1995; 34: 7668-7677Crossref PubMed Scopus (29) Google Scholar). This high fidelity could be achieved through the specific recognition by the synthetase of base sequences unique to the substrate tRNA, the singular solution structure of the tRNA, or both (8Hyde E.I. Eur. J. Biochem. 1986; 155: 57-68Crossref PubMed Scopus (7) Google Scholar). It is therefore necessary to characterize for every tRNA-synthetase system the roles of identity elements on the tRNA that are essential for recognition by the synthetase. NMR spectroscopy has been systematically applied to conformation analysis of tRNAs based on the finding that resonances from hydrogen-bonded GN1 and UN3 imino protons in RNA base pairs can be detected between 10 and 15 ppm in the 1The abbreviations used are: 2Dtwo-dimensional1Done-dimensionalTrpRStryptophanyl-tRNA synthetaseHSQCheteronuclear single quantum coherenceNOEnuclear Overhauser effectNOESYNOE spectroscopys4U4-thiouridine1The abbreviations used are: 2Dtwo-dimensional1Done-dimensionalTrpRStryptophanyl-tRNA synthetaseHSQCheteronuclear single quantum coherenceNOEnuclear Overhauser effectNOESYNOE spectroscopys4U4-thiouridine H NMR spectra, well resolved from other RNA protons that cluster between 3 and 9 ppm (9Moore P.B. Acc. Chem. Res. 1995; 28: 251-256Crossref Scopus (37) Google Scholar, 10Hare D.R. Reid B.R. Biochemistry. 1982; 21: 1835-1842Crossref PubMed Scopus (61) Google Scholar, 11Roy S. Papastavros M.Z. Redfield A.G. Biochemistry. 1982; 21: 6081-6088Crossref PubMed Scopus (34) Google Scholar, 12Hare D.R. Riberio N.S. Wemmer D.E. Reid B.R. Biochemistry. 1985; 24: 4300-4306Crossref PubMed Scopus (45) Google Scholar, 13Hyde E.I. Reid B.R. Biochemistry. 1985; 24: 4307-4314Crossref PubMed Scopus (19) Google Scholar). However, the assignments of the imino proton resonances remain problematic because of overlapping signals. Multidimensional NMR using stable isotopes such as 15N and 13C facilitates spectral assignments and the study of interactions between tRNAs and cognate synthetases (7Wallis N.G. Dardel F. Blanquet S. Biochemistry. 1995; 34: 7668-7677Crossref PubMed Scopus (29) Google Scholar, 14Niimi T. Kawai G. Takayanagi M. Noguchi T. Hayashi N. Kohno T. Muto Y. Watanabe K. Miyazawa T. Yokoyama S. Biochimie (Paris). 1993; 75: 1109-1115Crossref PubMed Scopus (6) Google Scholar). In a recent study, we found that designed mutagenesis of tRNA sequence provides a particularly powerful technique for the resolution of overlapping NMR signals in tRNA (15Yan X.Z. Xue H. Liu H.Z. Hang J. Wong J.T. Zhu G. J. Biol. Chem. 2000; 275: 6712-6716Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). By mutagenizing a base pair with a resonance that overlaps with that of another base pair, the latter resonance may be analyzed unambiguously. Use of this approach has made possible the assignment of almost all of the imino protons in the helical regions and the tertiary base pairs in Bacillus subtilis tRNATrp. two-dimensional one-dimensional tryptophanyl-tRNA synthetase heteronuclear single quantum coherence nuclear Overhauser effect NOE spectroscopy 4-thiouridine two-dimensional one-dimensional tryptophanyl-tRNA synthetase heteronuclear single quantum coherence nuclear Overhauser effect NOE spectroscopy 4-thiouridine Magnesium ions are essential to tRNA function, and their binding to tRNA has long been investigated (16Schimmel P.R. Redfield A.G. Annu. Rev. Biophys. Bioeng. 1980; 9: 181-221Crossref PubMed Scopus (111) Google Scholar). In tRNA molecules, weak nonspecific Mg2+ binding sites are abundant, primarily based on electrostatic interactions of the ion with backbone phosphates, and relatively weak in binding affinities. Strong binding sites are coordinated, either directly or via water, to phosphates and other ligands (17Jack A. Ladner J.E. Rhodes D. Brown R.S. Klug A. J. Mol. Biol. 1977; 111: 315-328Crossref PubMed Scopus (273) Google Scholar, 18Quigley G.J. Teeter M.M. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 64-68Crossref PubMed Scopus (360) Google Scholar, 19Westhof E. Sundaralingam M. Biochemistry. 1986; 25: 4868-4878Crossref PubMed Scopus (102) Google Scholar). Since the strong Mg2+ binding sites are non-randomly distributed and also few in number, their locations are expected to depend on RNA structure. The distributions of such strong Mg2+ sites therefore may furnish potentially useful structural information. Given the extensive imino proton assignments made possible by a combination of tRNA sequence mutagenesis and 2D1 NMR (15Yan X.Z. Xue H. Liu H.Z. Hang J. Wong J.T. Zhu G. J. Biol. Chem. 2000; 275: 6712-6716Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), the possible conformational roles of the A73 and G12C23 identity elements on bovine tRNATrp were examined in the present study based on their mutation to the ineffective forms of G73 and U12A23, respectively, and monitoring NMR chemical shift changes of different imino protons in the wild type and mutant molecules. Conformational changes were also detected through changes in the behavior of strong Mg2+ binding sites. Bovine tRNATrp (Fig. 1) was produced from hyperexpressing strains of E. coli JM109 transformed by recombinant pGEM-9Zf(−)-derived plasmid containing synthetic bovine tRNATrp gene and grown in M9-glycerol medium supplemented with 100 μg of ampicillin/ml (15Yan X.Z. Xue H. Liu H.Z. Hang J. Wong J.T. Zhu G. J. Biol. Chem. 2000; 275: 6712-6716Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The tRNATrp was purified as described by Xue et al. (20Xue H. Shen W. Wong J.T. J. Chromatogr. 1993; 613: 247-255Crossref PubMed Scopus (16) Google Scholar).15N-Labeled tRNATrp was obtained similarly except that NH4Cl was replaced by15NH4Cl (Isotec Inc.) in the growth medium. In addition to wild type bovine tRNATrp, the single base or base pair mutants of G73, G2C71, G27C43, and U12A23, in which the bases at positions 73, 2/71, 27/43, and 12/23 were changed to the designated forms, were also hyperexpressed, labeled, and purified. Tryptophanylation of the wild type and mutant tRNATrp was carried out with human TrpRS purified as described by Guo et al. (21Guo Q. Gong Q. Tong K. Vestergaard B. Costa A. Desgers J. Wong M. Grosjean H. Zhu G. Wong J.T. Xue H. J. Biol. Chem. 2002; 277: 14343-14349Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) using the assay method of Xue et al. (22Xue H. Shen W. Giege R. Wong J.T. J. Biol. Chem. 1993; 268: 9316-9322Abstract Full Text PDF PubMed Google Scholar). All NMR spectra were recorded on a Varian INOVA 500 spectrometer at a probe temperature of 30 °C. Jump-and-return sequence was applied in all 1D and 2D NOESY spectra for suppressing solvent signal as described by Yan et al. (15Yan X.Z. Xue H. Liu H.Z. Hang J. Wong J.T. Zhu G. J. Biol. Chem. 2000; 275: 6712-6716Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Phase-sensitive 2D NOESY spectra were recorded at a 120-ms mixing time with the hypercomplex method for quadrature detection in F1 dimension. A total of 256 t1 experiments with 4096 real points were collected over a spectral width of 12,000 Hz in each dimension. Sensitivity-enhanced gradient 2D 15N-1H HSQC spectra were recorded with a spectral width of 12,000 and 6000 Hz in the proton and nitrogen dimensions, respectively. One hundred and twenty-eight t1 increments were collected, each with 2048 real points. For NMR studies, 13–18 mg of tRNATrp as determined by UV absorbance at 254 nm was dissolved in 0.5 ml of Buffer A containing 10 mm MgCl2, 100 mmsodium chloride, and 10 mm sodium phosphate, pH 6.5. D2O was added to 8% as a lock signal. Magnesium ion was removed from the tRNA samples by dissolving the tRNA in 2 ml of Buffer A containing 100 mm EDTA in place of 10 mmMgCl2 and heating to 50 °C for 5 min. Afterward the solution was concentrated to 20% of the volume using Centracon-10 (Amicon Inc.), washed two to three times with the same EDTA-containing Buffer A, and washed three to four times more with the same buffer without EDTA. The final volumes of tRNA samples were adjusted to 0.45 ml. Titration with Mg2+ was achieved by adding successively small aliquots (5 μl) of a series of MgCl2 solutions of appropriated concentrations directly to the NMR tube. To construct binding curves of Mg2+ to tRNA, the chemical shifts of individual imino protons were plotted as a function of Mg2+concentration. Most data could be fitted to a one-binding-site model by means of software program Xcrvfit (Protein Engineering Network of Centres of Excellence (PENCE)/Medical Research Council of Canada (MRC) Group Joint Software Centre, Edmonton, Alberta, Canada). For binding curves with a clear departure from hyperbolic behavior in the form of a maximum, the data were fitted to a two-binding-site model by means of the same software (23Bradbury E.M. Nicolini C. NMR in the Life Sciences. Plenum Press, New York1986: 93-103Crossref Google Scholar). The usual assignment strategy for the imino proton resonances of nucleic acids was applied to the hydrogen-bonded segments of tRNA. The imino protons in these segments are close enough (≤5 Å) to give NOEs so that assignment of these protons can be achieved via a chain of connectivities provided a suitable starting point or a unique sequence is available (24Roberts G.C.K. NMR of Macromolecules: A Practical Approach. IRL Press, Cambridge1993: 217-288Google Scholar). Imino protons involved in hydrogen bonding in base pairs and tertiary interactions are protected from exchange with solvent and therefore visible in the downfield region of the 1H NMR spectrum (25Kearns D.R. Patel D. Shulman R.G. Yamane T. J. Mol. Biol. 1971; 61: 265-270Crossref PubMed Scopus (46) Google Scholar). Typically ∼28 such imino protons resonances are expected to appear for a canonical tRNA. For wild type bovine tRNATrp, 28 imino protons were distinguished in the region of the 1H NMR spectrum between 9 and 15 ppm (Table I). Two-dimensional 15N-1H HSQC (Fig. 2) and NOESY (Fig. 3) were both used for imino proton assignment. The 15N labeling allowed ready differentiation between UA (15N shifts 156.8–179.4 for UN3) and GC (15N shifts 142.9–148.2 for GN1) base pairs (7Wallis N.G. Dardel F. Blanquet S. Biochemistry. 1995; 34: 7668-7677Crossref PubMed Scopus (29) Google Scholar).Table IImino protons assignment of bovine tRNATrp wild type1H shift15N shiftAssignment1H shift15N shiftAssignmentppmppmppmppm14.81183.00s4U8-A1412.38148.06G42-C2814.24164.19U71-A212.35148.34G53-C6113.70164.43U52-A6212.19148.58G7-C6613.58160.95T54-A5811.87160.61U6513.22149.00G24-C1111.83159.52U513.16149.81G70-C311.77147.87G1-C7213.08163.09U29-A4111.65147.37G30-C4013.06149.43G22-C1311.64160.14—1-aThe resonance remained unassigned.12.96148.47G12-C2311.34161.04ψ55N312.91149.22G64-C5011.21146.42G4912.76149.73G67-C610.67158.77—1-aThe resonance remained unassigned.12.72148.72G69-C410.61145.00G6812.72150.23G10-C2510.34136.21ψ55N112.65148.70G51-C639.32147.13G181H and 15N chemical shifts of the imino protons from the HSQC spectrum of bovine tRNATrp wild type were recorded at 30 °C, pH 6.5 in H2O (8% D2O) with 10 mm sodium phosphate, 100 mm sodium chloride, and 10 mm magnesium chloride.1-a The resonance remained unassigned. Open table in a new tab Figure 3The low field region of the 2D1H NOESY spectrum of bovine tRNATrp wild type at 30 °C. All identified NOE cross-peaks at acceptor stem, D stem, anticodon stem, and T stem are connected by blue, black, green, and red straight lines, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 1H and 15N chemical shifts of the imino protons from the HSQC spectrum of bovine tRNATrp wild type were recorded at 30 °C, pH 6.5 in H2O (8% D2O) with 10 mm sodium phosphate, 100 mm sodium chloride, and 10 mm magnesium chloride. There is no special base in the acceptor stem that can be used as a starting marker for resonance assignment. To identify base pairs in this stem, a 15N-labeled tRNATrp mutant was prepared in which U71A2 was replaced by G2C71. The15N-1H HSQC spectrum of this G2C71 mutant indicated clearly that an imino proton resonance originally located at 14.24 ppm in the UA base pair region disappeared, accompanied by the emergence of a new resonance at 12.16 ppm in the GC base pair region. Based on this observation, the imino proton of U71 was unambiguously assigned. This imino proton gave NOEs to two different GC base pairs at 11.77 and 13.16 ppm, respectively. Since the base pair at 11.77 ppm was very weak in intensity, it was assigned to the imino proton of G1C72, which, being located at the open end of the acceptor stem, might be expected to undergo significant unstacking. The second base pair, which thereupon could be regarded as the imino proton of G70C3, gave rise to a further NOE to the G69C4 base pair. Because GU base pairs contain two hydrogen-bonded imino protons that are strongly dipolar-coupled on account of their close proximity (<3 Å), they usually yield the strongest cross-peaks in the NOESY spectrum (10Hare D.R. Reid B.R. Biochemistry. 1982; 21: 1835-1842Crossref PubMed Scopus (61) Google Scholar). On this basis the two GU base pairs in wild type bovine tRNATrp were therefore linked to the two strongest resonances in the upfield imino proton region. One of the base pairs gave NOEs to the identified G69C4, and the next GC base pair in the stem (namely G67C6) was assigned to G68U5. At the end of the acceptor stem, the imino proton of the G7C66 base pair was also connected in turn by NOE to the imino proton of G67C6. Thereby the base pairs in the acceptor stem were completely assigned. Like many other canonical tRNAs, bovine tRNATrp contains a sole ribothymidine residue at position 54. The thymidine methyl group resonates remarkably in the upfield region of 1H NMR spectrum at 0.99 ppm and is thus easily recognized. Since the proton of the methyl group is close to ψ55 and formed the reversed Hoogsteen pair T54A58 crossing the ribothymidine loop, it gave a set of NOE cross-peaks to their imino protons. This characteristic NOE crossing pattern in two-dimensional NOESY helped to assign the imino protons of T54 and ψ55 (15Yan X.Z. Xue H. Liu H.Z. Hang J. Wong J.T. Zhu G. J. Biol. Chem. 2000; 275: 6712-6716Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The identified T54 imino proton showed a further NOE connectivity to a GC base pair, assignable to the spatially adjacent G53C61. The imino proton of G53 in turn gave a very weak NOE to the AU base pair U52A62, which was confirmed in the15N-1H HSQC spectrum. The next NOE cross-peak between the imino protons of U52 and G51 could be traced out via T54/G53 and onward through G53/U52 NOE cross-peaks. Because there was no more distinct sequential NOE cross-peak available, the last two base pairs could be assigned only from the other end of the ribothymidine stem. Of the two GU base pairs in the tRNA, G68U5 in the acceptor stem was already assigned. Thus the remaining GU base pair could be identified unambiguously as G49U65. Based on the NOE cross-peak between G49U65 and the adjacent G64C50 base pair, the imino proton of G64 also could be assigned. The imino protons of G64 and G51 were too similar in chemical shift to display a distinguishable NOE peak from diagonal peaks. In addition to the three identified UA base pairs in the 15N-1H HSQC spectrum, only one characteristic UA base pair remained to be assigned at 13.08 ppm, which could be attributed unambiguously to U29A41 in the anticodon stem. The imino proton of this UA base pair gave NOEs to two GC base pairs at 12.38 and 11.65 ppm. No further sequential NOE connectivities related to these base pairs was observed. This might be due to the fact that since the UA and ψA base pairs are located at the two ends of the anticodon stem, dynamic fluctuations could cause them to become unstacked. To identify the GC base pairs in the anticodon stem, 15N-labeled mutant tRNATrpwith a base pair change from U27A43 to G27C43 was cloned, expressed, and purified. In the 15N-1H HSQC spectrum, a new GC imino proton resonance appearing at 13.16 ppm was assigned to this G27C43. A GC base pair originally located at 12.38 ppm in the wild type moved to 12.77 ppm in the G27C43 mutant, but no change in chemical shift was observed on another GC base pair located at 11.65 ppm. The 12.38 ppm GC base pair consequently could be assigned to the G42C28 base pair adjacent to U27A43, leaving the 11.65 ppm resonance assignable to G30C40. Another commonly encountered reversed Hoogsteen pair in tRNA, s4U8A14, provided an independent starting point for dihydrouridine stem assignments. Since the imino proton of this tertiary base resonated downfield to all other imino protons in both the 1H and15N dimensions on account of deshielding in thiouridine, it was readily identified at 15N shift 182.4 ppm. Based on the NOE between imino protons of s4U8A14 and its immediate neighbor of the G22C13 base pair (15Yan X.Z. Xue H. Liu H.Z. Hang J. Wong J.T. Zhu G. J. Biol. Chem. 2000; 275: 6712-6716Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), the G22 imino proton could be assigned to 13.06 ppm. However, no further NOE linked with G22C13 could be detected in the NOESY spectrum. This suggests that the G12/G22 NOE cross-peak was overlapped by diagonal signals because of the close similarity in chemical shifts between the imino protons of G22C13 and G12C23 base pairs. Three unidentified GC base pairs remained to be assigned in the 15N-1H HSQC spectrum. Two potential G12C23 base pairs resonated respectively at 12.96 and 13.22 ppm, yet another GC base pair at 12.72 ppm was connected to the 13.22 ppm base pair by NOE. To distinguish between these three GC base pairs, the15N-labeled U12A23tRNATrp mutant containing U12A23 in place of G12C23 was prepared and analyzed. In the 15N-1H HSQC spectrum of this U12A23 mutant, the wild type GC resonances at 12.96, 13.06, and 13.22 ppm, which would include that of G22C13, were missing from their original positions. Information from the NOESY spectrum suggests that the unaltered 12.72 ppm GC base pair could be assigned as G10C25, and the 13.22 ppm GC base pair to which G10C25 was linked by NOE could be assigned as G24C11. Since G22C13 at 13.06 ppm was already assigned based on its NOE to s4U8A14, the remaining GC base pair at 12.96 ppm was therefore assigned to G12C23. Besides the two assigned reversed Hoogsteen pairs of s4U8A14 and T54A58, wild type tRNATrp also contained the conserved tertiary ψ55G18 base pair between the T loop and D loop. The imino proton of G18resonated at 9.32 ppm, upfield from all other imino proton resonances, and gave an NOE to the ψ55N3 proton at 11.34 ppm, whereas the ψ55N1 proton was assigned on the basis of the mutual NOE at 10.34 ppm between N1 and N3 protons (26Quigley G.J. Rich A. Science. 1976; 194: 796-806Crossref PubMed Scopus (416) Google Scholar). The two possible UA resonances at 11.64 and 10.67 ppm in the 15N-1H HSQC spectrum, devoid of any detectable NOE connectivities, yet require further identification. Among the four 15N-labeled tRNATrp mutants G73, G2C71, G27C43, and U12A23, G73 gave 1D spectra most similar to the wild type. Most peaks in G2C71 also could be overlapped by those in wild type except for the evident loss of a UA resonance at 14.24 ppm and the emergence of a GC resonance at 12.16 ppm as expected from the UA to GC mutation. Both G27C43 and U12A23 underwent minor spectral changes, this being especially the case with the U12A23mutant (Fig. 4). Since the major spectral features of all these mutants largely resemble those of the wild type, all four mutant molecules must share with the wild type a closely similar three-dimensional conformation. The individual imino protons in the different mutant structures could be assigned simply by comparing the mutant and wild type 15N-1H HSQC spectra and combining the NOE information from their 2D NOESY spectra. This observation attests to the by and large replaceability of individual base pairs in tRNA with respect to the maintenance of the overall secondary and tertiary structures. It also underlines the straightforward and powerful use of tRNA mutants toward establishing NMR spectral assignments for tRNA molecules. Mg2+ binding curves for wild type tRNATrpand the G73 and U12A23 mutants were obtained by addition of Mg2+ to the tRNA molecules and monitoring the changes in the assigned imino proton chemical shifts. In the wild type and the G73 mutant, the imino protons of U65 and ψ55 in the T stem displayed the largest upfield shift changes followed by those of G67 of the acceptor stem, G53 of the T stem, and U29of the anticodon stem with larger downfield shifts (Fig. 5). These differences in magnitude of Mg2+-induced chemical shift changes could be the result of changes in the local environment of the proton or structural changes in the tRNA molecules (27Gonzales R.L., Jr. Tinoco I., Jr. J. Mol. Biol. 1999; 289: 1267-1282Crossref PubMed Scopus (79) Google Scholar). The U12A23 mutant displayed a similar pattern of chemical shift changes with additional large downfield shifts for the imino protons of s4U8 and G10 in the D stem. The majority of Mg2+ binding curves determined from the various assigned imino protons was hyperbolic and could be fitted to a one-binding-site model. Some of the curves, however, displayed a maximum and required fitting to a two-binding-site model (Table II). Because of the lack of a satisfactory procedure for calculating free Mg2+concentration in the face of multiple metal ion binding events (28Rüdisser S. Tinoco I., Jr. J. Mol. Biol. 2000; 295: 1211-1223Crossref PubMed Scopus (93) Google Scholar,29Maderia M. Hunsicker L.M. DeRose V.J. Biochemistry. 2000; 39: 12113-12120Crossref PubMed Scopus (63) Google Scholar), no attempt was made to estimate the exact Mg2+ binding dissociation constants. The fitted curves for imino protons associated with tight and medium Mg2+ binding sites are shown in Fig. 6.Table IIThe residues of bovine tRNATrp involved in the Mg2+bindingstRNATrpStrong binding (K1/2 of 1–10 mm)Medium binding (K1/2 of 10–20 mm)Weak binding (K1/2 of 20–500 mm)Marginal binding (K1/2 > 500 mm)WTG7,s4U8, G12,G24U52G1, U71, G70, G69, G10, G22, G18, G30, G42, U65, G64, G51, G53, T54, ψ55N3U5, G67, U29, ψ55N1G73G7,s4U8, G12,G24U52G1, U71, G70, G69, G10, G22, G18, G30, G42, U65, G64, G51, G53, T54, ψ55N3U5, G67, U29, ψ55N1U12A23s4U8, G24, G42U52, ψ55N3, ψ55N1G1, U71, G70, G69, G67, G10, G18, G30, U29, G42, U65, G64, G51, G53, T54G67, U29The residues whose imino proton require a two-binding-site model for description are shown in bold. The titration curves of the imino protons from U5, G67, U29, and ψ55N1 in wild type and G73 mutant and G67 and U29 in U12A23 mutant remained largely linear to high Mg2+ concentration, pointing to a very low affinity (>500 mm) interaction. The chemical shift changes of G68and G49 imino protons in wild type and G73 mutant and U5, G68, G22, and G49 in U12A23 mutant are too small to be used to estimate the K1/2. These residues therefore are not shown in the table. The imino protons of G12, G49, and G51are missing in the 15N-1H HSQC spectrum of U12A23 mutant; the K1/2 of these imino protons could not be measured. WT, wild type. Open table in a new tab The residues whose imino proton require a two-binding-site model for description are shown in bold. The titration curves of the imino protons from U5, G67, U29, and ψ55N1 in wild type and G73 mutant and G67 and U29 in U12A23 mutant remained largely linear to high Mg2+ concentration, pointing to a very low affinity (>500 mm) interaction. The chemical shift changes of G68and G49 imino protons in wild type and G73 mutant and U5, G68, G22, and G49 in U12A23 mutant are too small to be used to estimate the K1/2. These residues therefore are not shown in the table. The imino protons of G12, G49, and G51are missing in the 15N-1H HSQC spectrum of U12A23 mutant; the K1/2 of these imino protons could not be measured. WT, wild type. In both the wild type and G73 mutant, many of the imino protons conformed to hyperbolic titration curves of tight binding with a half-saturation Mg2+ concentration, or K1/2, of 1–10 mm, medium binding with K1/2 of 10–20 mm, weak binding with K1/2 of 20–500 mm, or marginal b" @default.
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• W2156294872 date "2002-06-01" @default.
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• W2156294872 title "NMR Analysis of Bovine tRNATrp" @default.
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