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J. Biol. Chem., Vol. 277, Issue 23, 20694-20701, June 7, 2002

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NMR Analysis of Bovine tRNA^Trp

CONFORMATION DEPENDENCE OF Mg²⁺ BINDING^*

Qingguo Gong, Qing Guo, Ka-Lok Tong, Guang Zhu, J. Tze-Fei Wong, and Hong Xue

From the Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China

Received for publication, March 8, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

NMR was used to study the solution structure of bovine tRNA^Trp 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 A⁷³ and the G¹²C²³ base pair on the D stem, two identity elements on bovine tRNA^Trp 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 U¹²A²³ mutant 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 tRNA^Trp. 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 U¹²A²³ mutant tRNA^Trp but not the wild type or G⁷³ mutant tRNA^Trp and that the G⁷, s⁴U⁸, G²⁴, and G²² imino 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 tRNA^Trp. 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 tRNA^Trp in the presence and absence of 10 mM Mg²⁺ indicated that overall tRNA^Trp structure stability was increased by more than 15 °C by the presence of Mg²⁺.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Although the three-dimensional structures of a number of free and enzyme-bound tRNA molecules have been elucidated with x-ray crystallography (1-6), 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 (7). 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 (8). 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 ¹H NMR spectra, well resolved from other RNA protons that cluster between 3 and 9 ppm (9-13). 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 (7, 14). 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 (15). 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 tRNA^Trp.

Magnesium ions are essential to tRNA function, and their binding to tRNA has long been investigated (16). 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 (17-19). 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 (15), the possible conformational roles of the A⁷³ and G¹²C²³ identity elements on bovine tRNA^Trp 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

tRNA^Trp Preparation and Assay-- Bovine tRNA^Trp (Fig. 1) was produced from hyperexpressing strains of E. coli JM109 transformed by recombinant pGEM-9Zf()-derived plasmid containing synthetic bovine tRNA^Trp gene and grown in M9-glycerol medium supplemented with 100 µg of ampicillin/ml (15). The tRNA^Trp was purified as described by Xue et al. (20). ¹⁵N-Labeled tRNA^Trp was obtained similarly except that NH₄Cl was replaced by ¹⁵NH₄Cl (Isotec Inc.) in the growth medium. In addition to wild type bovine tRNA^Trp, 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 tRNA^Trp was carried out with human TrpRS purified as described by Guo et al. (21) using the assay method of Xue et al. (22).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   The primary and secondary structure of bovine tRNATrp. The nucleotides bound tightly with Mg2+ determined in bovine tRNATrp are shown as black circles in wild type and G73 mutant and as bold letters in U12A23 mutant.

NMR Spectroscopy-- 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. (15). 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 tRNA^Trp as determined by UV absorbance at 254 nm was dissolved in 0.5 ml of Buffer A containing 10 mM MgCl₂, 100 mM sodium chloride, and 10 mM sodium phosphate, pH 6.5. D₂O was added to 8% as a lock signal.

Mg²⁺ Binding Curve-- 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 mM MgCl₂ 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 (23).

	RESULTS

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Imino Proton Resonances

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 (24). 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 (25). Typically ~28 such imino protons resonances are expected to appear for a canonical tRNA. For wild type bovine tRNA^Trp, 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 (7).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   The 2D 15N-1H HSQC spectrum of bovine tRNATrp wild type at 30 °C, pH 6.5 in H2O (8% D2O) with 10 mM sodium phosphate, 100 mM sodium chloride, and 10 mM magnesium chloride recorded on a Varian 500 MHz spectrometer. Twenty-six of the resonances shown have been assigned unambiguously and labeled accordingly. The two resonances marked by * remained unassigned.

Acceptor Stem-- 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 tRNA^Trp mutant was prepared in which U⁷¹A² was replaced by G²C⁷¹. The ¹⁵N-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 (10). On this basis the two GU base pairs in wild type bovine tRNA^Trp 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.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   The low field region of the 2D 1H 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.

Ribothymidine Stem-- Like many other canonical tRNAs, bovine tRNA^Trp 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 ⁵⁵ 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 ⁵⁵ (15). 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 the ¹⁵N-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.

Anticodon Stem-- 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 A 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 tRNA^Trp with 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.

Dihydrouridine Stem-- 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 and ¹⁵N 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 (15), 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, the ¹⁵N-labeled U¹²A²³ tRNA^Trp 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 tRNA^Trp also contained the conserved tertiary ⁵⁵G¹⁸ base pair between the T loop and D loop. The imino proton of G¹⁸ resonated at 9.32 ppm, upfield from all other imino proton resonances, and gave an NOE to the ⁵⁵N3 proton at 11.34 ppm, whereas the ⁵⁵N1 proton was assigned on the basis of the mutual NOE at 10.34 ppm between N1 and N3 protons (26). 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 tRNA^Trp 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 U¹²A²³ mutant (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.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   The 1D imino proton NMR spectra of bovine wild type (a), G73 (b), G2C71 (c), G27C43 (d), and U12A23 (e) at 30 °C.

Magnesium Ion Binding

Mg²⁺ binding curves for wild type tRNA^Trp and 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 ⁵⁵ 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 U²⁹ of 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 (27). 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.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   The maximum range of chemical shift changes of individual imino protons in imino proton titration experiments of bovine tRNATrp wild type and two mutants (G73 and U12A23) with Mg2+. The experimental data of U12, G49, and G51 are missing for mutant U12A23 because corresponding imino protons cannot be found in 2D 15N-1H HSQC spectrum.

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 (28, 29), 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.

View this table: [in this window] [in a new window]   Table II The residues of bovine tRNATrp involved in the Mg2+ bindings 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 55N1 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 G68 and 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 G51 are missing in the 15N-1H HSQC spectrum of U12A23 mutant; the K1/2 of these imino protons could not be measured. WT, wild type.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Imino proton titration studies of bovine tRNATrp wild type and two mutants (G73 and U12A23) with Mg2+. The sample conditions are 10 mM sodium phosphate, 100 mM NaCl, pH 6.5 at a temperature of 30 °C. The binding curves of chemical shift versus Mg2+ concentration for several typical imino protons are shown by fitting the data to a one- or two-binding-site model.

Wild Type and G⁷³ Mutant-- 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 K_1/2, of 1-10 mM, medium binding with K_1/2 of 10-20 mM, weak binding with K_1/2 of 20-500 mM, or marginal binding with K_1/2 of >500 mM (Table II). The chemical shift changes of G⁶⁸ and G⁴⁹ imino protons were too small to give accurate binding curves, whereas U⁵² in T stem and G¹² in D stem displayed exceptionally strong binding (Fig. 6). On the other hand, the Mg²⁺ binding curves of the G⁷, s⁴U⁸, G²⁴, and G²² imino protons were obviously non-hyperbolic and thus required fitting by a two-binding-site model. Among them, G⁷, s⁴U⁸, and G²⁴ exhibited strong binding of the first magnesium ion with low K_1/2 values in the range of 1-10 mM (Fig. 6).

U¹²A²³ Mutant-- The U¹²A²³ mutant displayed a number of differences from the wild type. Its imino protons from U¹², G⁴⁹, and G⁵¹ were unexpectedly missing, probably overlapped by other resonances in the ¹⁵N-¹H HSQC spectrum. Remarkably, besides its imino protons of U⁵, G⁶⁸, G⁷, and G²², which scarcely changed their chemical shifts during the Mg²⁺ titration, the titration curves of all its observed imino protons were hyperbolic and could be fitted by the one-site model. This behavior of the U¹²A²³ mutant was a sharp departure from the wild type or G⁷³ mutant in which the response of four imino protons to Mg²⁺ binding required a two-binding-site model for description. While the s⁴U⁸ and G²⁴ imino protons in U¹²A²³ remained associated with tight Mg²⁺ binding, the U⁵² imino proton in the ribothymidine stem and G⁴² imino proton in the anticodon stem were indicative of much stronger Mg²⁺ binding compared with the wild type and G⁷³ molecules (Fig. 6). Similarly an increase in Mg²⁺ binding affinity also was evident with ⁵⁵N1 and ⁵⁵N3, which were marginal sites of Mg²⁺ binding in both wild type and G⁷³ mutant.

Temperature Effects

The ¹H NMR spectra of wild type bovine tRNA^Trp in the presence or absence of Mg²⁺ at different temperatures are shown in Fig. 7. In the presence of 10 mM Mg²⁺, most of the peaks changed their chemical shifts upfield with increasing temperature and gave indications of melting with peak intensity reduction and signal broadening. Comparing the 2D ¹⁵N-¹H HSQC spectra obtained at different temperatures, the signals due to U⁷¹ and U⁵² were found to weaken at 40 °C and almost vanish at 50 °C. The signal of ⁵⁵N1, which is not hydrogen-bonded, became broadened at 40 °C and totally disappeared at 60 °C. The peak intensities of G¹ and G¹⁸ decreased sharply at 50 °C and disappeared at 60 °C. The peaks of U²⁹ and G³⁰ in the anticodon stem displayed reduced intensities at 50 °C. Almost all resonances underwent considerable reduction in intensity and peak broadening at 60 °C, especially those of the two GU base pairs G⁴⁹U⁶⁵ and G⁶⁸U⁵. The total melting of tRNA structure at 70 °C was indicated by the disappearance of all imino resonances.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   1D imino proton NMR spectra of bovine tRNATrp wild type at different temperatures in the presence (left) and absence (right) of 10 mM magnesium ions.

These observations suggest that melting began at 40 °C at the 3'-end of acceptor stem and the U⁵²A⁶² region of T stem and gradually spread to the entire anticodon stem, the acceptor stem, and the T stem. That only NOE connectivities of the D stem remained observable in 2D NOESY at 50 °C points to a higher degree of stacking in the D stem, thus resisting against extensive melting up to 70 °C. The tertiary interaction between ⁵⁵ in T loop and G¹⁸ in D loop was disrupted completely at 60 °C, whereas the other two tertiary base pairs s⁴U⁸A¹⁴ and T⁵⁴A⁵⁸ did not vanish until 70 °C. Thus the tertiary base pair G¹⁸⁵⁵, being located at the outer corner of the L-shaped structure of the tRNA molecule (1, 2), evidently became more readily accessible to solvent exchange with increasing temperature.

In the absence of Mg²⁺, the imino proton peaks of the tertiary interactions s⁴U⁸A¹⁴ and G¹⁸⁵⁵ were already diminished at 30 °C. At 40 °C, most of the imino proton signals lost substantial intensities. However, the U⁷¹, G⁷⁰, G⁶⁹, U⁵, and G⁶⁸ peaks in the acceptor stem, although reduced, were clearly visible. Although the tertiary interactions and the other stems melted by 50 °C, the acceptor stem remained largely intact. By 60 °C, all the imino proton signals in the tRNA^Trp disappeared (Fig. 7, left and right).

Tryptophanylation Activity

Previously it was shown that the discriminator base N⁷³ is an important element on tRNA^Trp toward the productive catalytic recognition by TrpRS with bacterial TrpRS preferring the presence of G⁷³ and eukaryotic and archaeal TrpRS preferring the presence of A⁷³ (21, 22). As shown in Fig. 8, mutation of A⁷³ on wild type tRNA^Trp to G⁷³ brought about a steep decrease in tryptophanylation by human TrpRS, and mutation of G¹²C²³ to U¹²A²³ also brought about a significant decrease. The observation confirmed A⁷³ as a major identity element and G¹²C²³ as a minor identity element on bovine tRNA^Trp.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Comparative tryptophanylation efficiency of bovine tRNATrp wild type (WT) and U12A23 and G73 mutants by human TrpRS (2 nM).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Through ¹⁵N labeling in vivo with the use of different mutants to remove overlapping resonances, the resonances of all 26 observable imino protons participating in secondary and tertiary base pairings in bovine tRNA^Trp hyperexpressed in E. coli have been assigned. By providing another example in addition to the earlier study of B. subtilis tRNA^Trp (15), these findings establish the generality of this powerful experimental approach for characterization of tRNA structures by the combination of NMR and sequence mutagenesis. They also made possible an analysis of the relationship between Mg²⁺ binding sites and tRNA conformation.

Mg²⁺ Binding Sites-- NMR spectroscopic studies of the wild type and mutant tRNA^Trp, moreover, made possible a dissection of the Mg²⁺ binding sites. Mg²⁺ is known to strongly stabilize the native tertiary structures of tRNA even in the presence of substantial univalent salt concentrations (30), and a great deal of effort has been made to understand the Mg²⁺ binding to tRNA.

The crystallographic structure of yeast tRNA^Phe, in which coordinated magnesium ions were first identified, has provided a valuable basis for the thermodynamic analysis of Mg²⁺ binding (31). Based on temperature factors (B factors) of the crystal structure, there are four strong Mg²⁺ binding sites located on the D stem and D loop and six weaker sites distributed over the four stems and the anticodon loop. This suggests that the Mg²⁺ binding depends on specific localized structure on the tRNA, and the important question arises regarding the relationship between Mg²⁺ binding and tRNA conformation. Since NMR is particularly useful for the investigation of macromolecular structures, in the present study NMR analysis of wild type and mutant tRNA^Trp was performed to address this question.

Previously Mg²⁺ binding to tRNA^Gly, tRNA^Phe, tRNA^Val, and mitochondrial tRNA^Ser was investigated using ¹H NMR spectroscopy (8, 32-35). Delineation of different Mg²⁺ binding sites was limited, however, due to inadequate resolution and assignment of the imino protons in the 1D NMR spectra. In contrast, assignment of all 26 observable imino protons in the stems and tertiary base pairs of bovine tRNA^Trp rendered straightforward the titration of chemical shift changes of individual imino protons as a function of Mg²⁺ concentration. On this basis, in wild type tRNA^Trp G⁷, s⁴U⁸, G¹², and G²⁴ were found to be associated with strong Mg²⁺ binding, U⁵² with medium binding, and 19 other protons with weak binding. Moreover, in so far that the Mg²⁺ titration curves of the G⁷, s⁴U⁸, G¹², and G²² proton exhibited a clear maximum, their behavior requires fitting to a two-binding-site model rather than a one-binding-site model of Mg²⁺ binding (Fig. 5 and Table II). The responses of imino protons to Mg²⁺ addition in the G⁷³ mutant tRNA^Trp are closely similar to those observed in the wild type. The maximum chemical shift change induced by the addition of magnesium ions varies with the imino protons (Fig. 5). It is noteworthy that the maximum chemical shift change is not tightly correlated with the strength of Mg²⁺ binding. For example, large chemical shift changes were displayed by ⁵⁵N1, U⁶⁵, and G⁶⁷ protons, and small chemical shift changes were displayed by G¹⁸ and U⁵, yet all five of these protons were associated with loose Mg²⁺ binding. This is not entirely surprising. Since chemical shift changes are usually dependent on the extent of chemical environmental alteration and ring current variations associated with individual nucleotides (27, 36), these parameters are not expected to be correlated exactly with the thermodynamics of Mg²⁺ binding.

Mutagenesis of the G¹²C²³ base pair in the wild type to U¹²A²³ altered the mode of Mg²⁺ binding at the D stem and nearby region. Both s⁴U⁸ and G²⁴, affected by two molecules of Mg²⁺ in the wild type, became associated with a single-site Mg²⁺ binding mode (Fig. 6). Furthermore, the Mg²⁺-induced chemical shift change in s⁴U⁸ in the mutant molecule over the higher Mg²⁺ concentration above 2 mM were opposite in direction to the change in wild type. Such behavior indicates strongly the occurrence of important conformation change in the D stem region upon mutation of G¹²C²³ to U¹²A²³.

Thermal Stability-- The binding of Mg²⁺ to the various binding sites on tRNA^Trp resulted in evident stabilization of both secondary and tertiary structures. In the absence of Mg²⁺, base pairs in the acceptor stem retained at 50 °C visible resonances even with all the tertiary interactions disrupted and the three other stems largely melted. This is to be expected given the seven base pairs, as many as five of these GC, in the acceptor stem. In the presence of 10 mM Mg²⁺, however, the 2D ¹⁵N-¹H HSQC results suggest that the D stem was the most stable element in the molecule followed by T stem and acceptor stem with the anticodon stem having the lowest stability. Overall tRNA^Trp structure stability was increased by more than 15 °C upon the addition of 10 mM Mg²⁺. The exceptional stability of the D stem, with only four base pairs, in the presence of Mg²⁺ is entirely consistent with the Mg²⁺ binding sites delineated in Table II. Of the four nucleotide residues associated with strong Mg²⁺ binding, G⁷ is located on the acceptor stem next to the junction with D stem, while the s⁴U⁸A¹⁴ base pair spans the D stem. G¹² and G²⁴ both form part of the D stem itself. This very special relationship between the D stem and strong Mg²⁺ binding in all likelihood may be important rivets contributing to the remarkable stability of the D stem in the presence of 10 mM Mg²⁺.

As shown in Fig. 8, A⁷³ on bovine tRNA^Trp functions as a major identity element, and G¹²C²³ functions as a minor identity element. Mutation of these elements to G⁷³ and U¹²A²³, respectively, decreases tryptophanylation activity. Mutation of A⁷³ to G⁷³ brought about minimal alterations with respect to either NMR spectrum (Fig. 4) or Mg²⁺ binding sites (Fig. 6). In contrast, mutation of G¹²C²³ to U¹²A²³ brought about evident alteration in the NMR spectrum (Fig. 4) as well as the Mg²⁺ binding mode of s⁴U⁸ and G²⁴, attesting to the occurrence of conformational change in the U¹²A²³ mutant molecule. Therefore A⁷³ as an identity element is most likely recognized directly by TrpRS. G¹²C²³ as an identity element, on the other hand, evidently contributes to productive TrpRS-tRNA^Trp recognition at least in part through the maintenance of an optimal conformation that was affected by mutation to U¹²A²³.

The Mg²⁺ binding sites mapped by NMR in this study are in agreement with the x-ray crystallography results (31) in that both point to the clustering of strong Mg²⁺ binding sites in the D stem and its junctions with the acceptor stem and anticodon stem. Thus NMR analysis when combined with sequence mutagenesis has made possible extensive assignment of imino proton resonances, which in turn has permitted the mapping and characterization of multiple Mg²⁺ sites on the tRNA. Once mapped and characterized, these sites provided an array of conformational markers that are very sensitive to conformational change in the tRNA molecule. This will open up the way for proving the relationship between tRNA sequence and conformation as well as the sequences and conformations optimal for recognition by cognate aminoacylation-tRNA synthetases.

	FOOTNOTES

^* This study was supported by the Research Grant Council of Hong Kong.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 852-2358-8707; Fax: 852-2358-1552; E-mail: hxue@ust.hk.

Published, JBC Papers in Press, March 27, 2002, DOI 10.1074/jbc.M202299200

	ABBREVIATIONS

The abbreviations used are: 2D, two-dimensional; 1D, one-dimensional; TrpRS, tryptophanyl-tRNA synthetase; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; s⁴U, 4-thiouridine.

	REFERENCES

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