A 15N-1H nuclear magnetic resonance study on the interaction between isoleucine tRNA and isoleucyl-tRNA synthetase from Escherichia coli

Biochimie (1993) 75, 1109-1115

© Soci6t6 fran~aise de biochimie et biologic mol6culaire/ Elsevier, Paris

1109

A mSN-1Hnuclear magnetic resonance study on the interaction between isoleucine tRNA and isoleucyl-tRNA synthetase from Escherichia coli T N i i m i a, G K a w a i b,c, M T a k a y a n a g i b, T N o g u c h i b, N H a y a s h i d, T K o h n o a**, Y M u t o a, K W a t a n a b e c, T M i y a z a w a b***, S Y o k o y a m a a* aDepartment of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113; bFaculty of Engineering, Yokohama National University, Tokiwadai, Hodogaya-ku, Yokohama 240; cDepartment of Industrial Chemistry, Faculty of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113; dDepartment of Biological Science, Tokyo hzstitute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 22 7, Japan (Received 19 November 1993; accepted 29 December 1993)

Summary - - Imino 15N and IH resonances of Escherichia coli tRNA[le were observed in the absence and presence of E coli isoleucyl-tRNA synthetase. Upon complex formation of tRNA]le with isoleucyl-tRNA synthetase, some imino 15N-IH resonances disappeared, and some others were significantlybroadened and/or shifted in the IH chemical shift, while the others were observed at the same 15N-IHchemical shifts. It was indicated that the binding of tRNA[le with IIeRS affect the following four regions: the anticodon stem, the junction of the acceptor and T stems, the middle of the D stem, and the region where the tertiary base pair connects the T, D, and extra loops. This result is consistent with those of chemical footprinting and site-directed mutagenesis studies. Taken together, these three independent results reveal the recognition mechanism of tRNA]Ieby IleRS: IIeRS recognizes all the identity determinants distributed throughout the tRNA[le molecule, which induces changes in the secondary and tertiary structures of tRNA[le . tRNA / aminoacyl.tRNA synthetase ! isotope labeling / NMR

Introduction The correct interpretation of the genetic code requires each of the twenty members of the ARS family to strictly recognize its cognate tRNA(s) and amino acid. Recently, several cases have been found where the amino acid specificity of the tRNA depends on a relatively small number of nucleotide residues (identity determinants) [1-7]. For most tRNAs, nucleotide residue(s) in the anticodon and, in several cases, the 'discriminator base' at position "13 are the dominant identity determinant(s) [ 1-9]. In the acceptor stem, the

*Correspondence and reprints **Present address: Mitsubishi Kasei Institute of Life Sciences, Minamiooya, Machida, Tokyo 194, Japan. ***Deceased. Abbreviations: acp3U, 3-(3-amino-3-carboxypropyl)ufidine; ARS, aminoacyl-tRNA synthetase; D, dihydrouridine; HMQC, heteronuclear multiple quantum coherence spectroscopy; IIeRS, isoleucyl-tRNA synthetase; mTG, 7-methylguanosme; 2-D, two-dimensional; T, ribothymidine; t6A, N-((9-~-D-ribofuranosylpurin-6-yl)carbamoyl)threonine;W, pseudouridine.

base pairs 1:72, 2:71, and/or 3:70 are major or minor determinants of several tRNAs [1-9]. The threedimensional structures of the complexes of E coli tRNAOln and yeast tRNAAsp with their cognate ARSs have been elucidated by X-ray crystallography [10-12]. These ARSs recognize the identity determinants in the anticodon loop and the 5' and 3' terminal regions (the two ends of the L-shaped structure) of the tRNA [13, 14], resulting in local conformational changes in these regions [ 10--12]. In other regions, however, only a few identity determinants have been found [14-16]. As for E coli tRNAPhe, tRNASer, tRNALeu etc, a number of residues in the central region of the L-shaped structure have been reported to be important for the aminoacylation, although it is not likely that all of them are identity determinants [1, 17, 18]. This is probably because conformational aspects of these tRNAs are essential for their aminoacylation activities. Therefore, it may be necessary to inspect the entire L-shaped structure containing the secondary and tertiary base pairs of tRNA. For this purpose, it is useful to perform NMR analyses of hydrogen-bonded imino proton reso-

1110 145 0 G49

G40 G50 G53/ GI5 G39 G24G3\G2~/--]~ I~ ~ oi 0 fixa0~ [ ' ~ G52 G7

G43

G68 0

150

G2 GI0 m7G46

155,_. Z 160

W65N3

u, j4

0

us d

0 UI2 O, t US! 0 d6 U42 U8

~55N3

165

170 15

1'4

1'3tH (ppm)12

11

10

Fig 1. 15N-IH 2-D HMQC spectrum of uniformly ,SNlabeled tRNA~te. Cross-peaks are labeled with the residue • assignments. The W 55N 1H peak lies outside of the plotted region. The cross-peak labeled with an asterisk is due to contamination (no ~H peak was observed at this chemical shift for unlabeled tRNA~Ie[27]). nances. In particular, the ~SN-~H correlation method greatly helps the analysis of the tRNA solution structure [19-24]. Although it is much harder to apply this method to larger molecules, Redfield et al have studied the interaction of tRNACtn and its cognate ARS from E coli [25]. As for E coil tRNAlle, we have elucidated the identity determinant elements located at the anticodon loop and stem, the D stem, the acceptor stem, and the CCA terminus by analyses of chemical footprinting and aminoacylation kinetics of tRNA variants [26]. Furthermore, it has been suggested that tRNA]lc undergoes a global conformational change upon binding with IIeRS [26]. In the present study, on the basis of the previous assignment of the hydrogenbonded imino proton resonances of tRNA~I¢ [27], we identified the regions that were affected in the complex formation with IleRS by the tSN-~H correlation method. These results were strikingly consistent with those of the footprinting and kinetics studies. Thus, the combination of these three different analyses uncovered the recognition mechanism of tRNAII~ by IIeRS.

Materials and methods

Preparation of lleRS and/SN-labeled tRNA~tefi.om E coli E coli IIeRS was prepared as described [26, 28, 29]. Uniformly 15N-labeled tRNA was prepared from E coli Ai9 cells cultured

in a minimal medium containing 15NH4CI (99.9 Atom% 15N, Isotec Inc) [ 19].lSN-Labeled tRNA[te was obtained by two-step chromatography on column of Q-Sepharose HP (Pharmacia) using a Bio-HPLC system (Tosoh, Tokyo). N M R measurements

All samples were dissolved in H20 (pH 6.7) containing 100 mM NaCI, 10 mM MgCI2, and 5% 2H20. NMR spectra were recorded on Bruker AMX-500 spectrometers at a probe temperature of 37°C. The 2-D HMQC experiments [30] were carded out with an interpulse delay time of 4.2 ms, which was optimized experimentally (to be published elsewhere). The tRNA concentration for tRNA]Ie in the free state was 0.40 mM. For the 2-D HMQC spectra of tRNA~le in the IleRS-bound state, a complex of tRNA~le and unlabeled IIeRS (0.40 mM and 0.65 mM, respectively) was prepared. The jump-and-return pulse [31] was used for suppression of the water resonance. The spectral width for IH was 12000 Hz and that for 15N was 2000 Hz. The spectra were collected in the phase-sensitive mode, using the time-proportional phase incrementation method [32]. Free induction decays (256 scans each) of 4096 real data points in the t2 domain were collected for 256 data points in the t, domain. By zero-filling in the t~ domain and 90°-shifted squared-sine-bell apodization in both the tl and t2 domains followed by Fourier transformation, spectra of 4096 x 512 data points were obtained. The chemical shifts for IH and 15N were referenced to the methyl proton resonance of sodium 2,2-dimethyl-2-silapentane-5-sulfonate and to the resonance of 15NH4CI, respectively. It was confirmed by electrophoresis that the tRNA0e in the mixture with the purified IIeRS was not degraded during the course of the NMR measurements.

Results and discussion

Effects of complex ~brmation on base pairs of tRNAt/e Figure 1 shows the 2-D 15N-~H H M Q C spectrum of the uniformly 15N-labeled tRNA[re. On the basis of the previous assignment of the imino proton resonances of tRNA]te [27], the assignment of the 15N-IH crosspeaks, due to all the secondary base pairs and the seven tertiary base pairs, was accomplished (fig 1, table I). These results confirmed the previous assignment. Moreover, the imino proton resonating at 11.55 ppm was identified to be N3H rather than N IH of 65, because the irradiation of this imino proton produced no sharp NOE peak in the aromatic proton region where C6 protons resonate (data not shown). In the 2-D H M Q C spectrum of the complex of tRNA[le and IIeRS (fig 2), most of the 15N-,H crosspeaks of the tRNA{~e in the IleRS-bound state were still observed at the same chemical shifts as those of the ~SN-~H cross-peaks of the tRNA]le in the free state, and were appreciably broadened. As a representative example, the cross-peak of G49-q j 65(N3H) is shown in figure 3a (the observed residue is indicated in bold letters). However, a limited number of cross-peaks were significantly affected upon binding with IIeRS. The cross-peaks of U6-A67 and C29-G41 were

1111 shifted by 0.04 ppm and 0.08 p p m on the IH axis, respectively. The heights of the cross-peaks of C I 1G24, U12-A23, and G7-C66 decreased far more than those of the others observed upon binding with IIeRS, because of the broadening of the imino proton resonances (the cross-peak of U12-A23 is shown in fig 3b). Furthermore, the cross-peaks of C30-G40, G19C56, G15-C48, C31-G39, G49-W65, and U 5 - G 6 8 became unobservable in figure 2 (the cross peak of G49-~F 65 is shown in fig 3c). By ~H-NMR analysis, these imino proton resonances have disappeared upon complex formation [27]. The chemical shifts of the 15N-IH cross.peaks of tRNA~ le bound with IIeRS are listed in table I.

Regions that were affected upon binding with IIeRS TLe base pairs with imino proton resonances that were affected upon binding with IIeRS are indicated in the L-shaped structure of tRNA[ le (fig 4). The binding of this tRNA with IIeRS is thus found to affect the following four regions of tRNA[~e: the anticodon stem, the junction of the acceptor and T stems, the middle of

145

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1.17~$4 *

*

160-

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U42

170

15

1'4

131H (ppm)I2

In the absence of lleRS (chemical shift) (ppm) IH tSN base pair 14.39 14.15 13.85 13.80 13.73 13.71 13.67 13.45 13.35 13.25 13.22 13.16 13.05 12.95 12.94 12.76a 12.76a 12.76a 12.68 12.60 12.33 12.13 12.00 11.78 I 1.77 11.55 11.40 1 I. 19

U5~F~N3 IJ6

Table I. Chemical shifts of imino IH and 15N resonances of secondary and tertiary base pairs of tRNA~le in the absence and presence of IleRS.

I'I

10

Fig 2. 15N-IH 2-D HMQC spectrum of the complex of uniformly 15N-labeled tRNA~ie and IIeRS. Cross-peaks are labeled with their assigned residues when the corresponding cross-peaks are observed in figure 1. For the underlined residues, the shifts of the imino proton resonances upon lleRS-binding were larger than 0.03 ppm. For the boxed residues, the broadening of the imino proton resonances was more than for other observed ones (see text). Asterisks denote resonances due to contamination. The levels of the contours in the plots is not the same as that in figure 1.

10.90 10.41

In the presence of lleRS (chemical shift) (ppm) tH t5N typed

166.9 U8-A14b " 14~39 166.9 165.8 U6-A67 14.11 165.8 S 163.5 U12-A23 13.85 163.5 B 165.4 A28-U42 13.80 165.4 162.7 A1-U72 13.73 162.8 151.3 Cl1-G24 13.70 151.3 B 162.6 T54-A58b 13.67 162.7 164.8 U51-A63 13.45 164.7 151.6 C4-G69 13.35 151.7 151.7 G3-C70 13.26 151.7 154.2 C27-G43 13.22 154.4 151.2 G50-C64 13.15 151.3 151.5 C29-G41 13.13 151.5 S 150.9 C13-G22 12.96 151.1 150.9 G53-C61 12.96 151.1 150.5 G2-C71 12.77 150.8 150.5 G10-C25 19.75 150.8 151.8 G22-mTG46b 12.75 152.0 150.7 C30-G40 U 150.8 G19-C56 b U 150.2 G15-C48 b U 150.0 G52-C62 12.15 150.2 149.9 C31-G39 U 150.1 G7-C66 11.78 150.3 B 161.6 US-G68 c 11.78 161.5 160.6 G49-~ 65(N3H)c 11.57 160.7 162.7 W 55(N3H) b 11.40 162.7 147.0 G49-W 65 c U 146.6 U5-G68 c U 137.4 ~ SS(NIH) b 10.40 137.8

aln the 2-D HMQC spectrum, two of these three cross-peaks were overlapped with each other, bTertiary base pair. cWobble base pair. dTypes of effects of IleRS-binding on the imino proton resonance: S, shifted more than 0.03 ppm; B, broadened more than other observed ones; U, unobservable in the complex with IIeRS. the D stem, and the region where the tertiary base pairs connect the T, D, and extra loops.

The anticodon stem In the anticodon stem, the imino proton resonance of C29-G41 was shifted and those of C 3 0 - G 4 0 and C31

1112

a

b

c

~5N=160.6 ppm

~SN=163.5 ppm

lSN=147.0 ppm

is N=160.7 ppm

Is N= 163.5 ppm

Is N= 147.0 ppm

I

I

12.0

11.5 ppm

I

I

11.0 14.5

I

14.0 ppm

I

I

13.5 11.5

I

11.0 ppm

I 10.5

Fig 3. The cross-sections along the o~_(IH) dimension from the 2-D HMQC spectra of uniformly 15N-labeledtRNA{le (top) and of the complex of uniformly 15N-labeled tRNA~le and IIeRS (bottom). 15N chemical shifts are 160.6 ppm and 160.7 ppm (a), 163.5 ppm (b) and 147.0 ppm Ic). G39 disappeared upon binding with IleRS. The disappearance of the imino proton resonances of the base pairs upon binding of tRNA[le with IIeRS is likely to arise from rapid proton exchange with the solvent water. There are two possible mechanisms of rapid solvent exchange of imino protons [35]. First, some basic residues of IIeRS could approach the imino proton of the base pair of tRNA[le and catalyze its exchange. Second, the hydrogen bonds are disrupted, either continuously or discontinuously, and the imino proton of the base pair is exposed to the solvent. In either case, such rapid exchange must arise from conformational changes around the base pairs, because the secondary and tertiary base pairs of the Lshaped tRNA molecule are firmly stacked upon one another, with only a few exceptions, and therefore the imino protons cannot easily be accessed by either the protein or the solvent. Therefore, the large effects on the consecutive base pairs in the anticodon stem upon binding with IIeRS indicate that this region undergoes a significant conformational change, such as an unwinding of the stem, In this context, by the kinetic analysis, it was shown that the anticodon residues and G41 in the middle of the anticodon stem are the major

identity determinants of tRNAI~e [26]. Furthermore, by the chemical footprinting analysis, the anticodon loop and the 3'-strand of the anticodon stem were shown to be in contact with IIeRS in the complex [26]. Taken together, these results suggest that IleRS recognizes the anticodon residues and G41 by direct interaction with the anticodon loop and the 3'-strand of the anticodon stem of tRNA[~e, which induces the conformation change at the anticodon stem. Junction of the acceptor and T stems In the junction of the acceptor and T stems, the imino proton resonance of U6-A67 was shifted and that of G7-C66 was more broadened than the other obse~ed resonances upon binding with IIeRS. Interestingly, the imino proton resonances of the guanine residues in the wobble base pair (G49-W 65 and U5-G68) disappeared, while those of uridine residues were not affected. Thus, upon complex formation, these wobble base pairs appear to be partially disrupted or distorted at the minor groove. It has been found that the C4G69 base pair and A73 are included in the identity determinants and that the phosphate groups at positions 70 and 72 are in contact with IIeRS [26]. There-

1113 fore, it was suggested that IIeRS binds with the CCA terminus and the top of the acceptor stem, and induces conformational changes in the junction region. These are relatively small and probably represent a slight distortion of the base pairs.

has been found to be an identity determinant and to interact with IIeRS [26], it was likely that IIeRS recognizes this base pair, resulting in the distortion of the helix structure in the D stem.

Global conformational change upon binding with lleRS The imino proton resonances of G19-C56 and G15C48 disappeared upon binding with IIeRS. G19-C56 connects the T and D loops, while G 15-C48 connects the D and extra loops (fig 4). Intriguingly, it has been shown that these two base pairs are dispensable for specific aminoacylation [26]. Furthermore, chemical modification analysis has indicated that the interaction between the T and D loops of tRNA]~e is disrupted in the complex [26]. In contrast, the imino proton resonances of U8-A14 and G22-mTG46 were not affected upon binding with IIeRS. Correspondingly, these two base pairs have been found to be essential for the tRNA~l~ activity [26]. Therefore, it was indicated that some specific tertiary base pairs involving G19-C56 and G 15-C48 are disrupted, and concomitantly, the Lshaped structure changes upon complex formation. This global conformational change appears to be important for specific aminoacylation of tRNA[~e. Taken together, these results uncovered the recognition mechanism of tRNA]le by IIeRS: IleRS recognizes not only the two ends of the L-shaped structure (the anticodon loop and the 3' terminus), but also several base pairs at the stems, which induces the local conformational changes at the three stem regions, and furthermore, the global conformational changes of tRNA]Ie.

The D stem In the D stem, the imino proton resonances of C I 1G 2 4 and U12-A23 were more broadened than the other observed ones. Because the base pair U12-A23

a G 60 65 70 A.. UCCAC~F_C A G G C C U A C C A C ".,,,GGUGG G U U C G G A

G..C m7.GI A[0 ................I A GIC 456 I

25 C ' G lo

G C.G A.U CBG

30 g i g

I

'

A I

40

cCIGA U t6A GAU 35

Comparison with other tRNA-ARS systems

b

As for E coli tRNArMet, it has been shown by ribonuclease footprinting analysis that the tertiary interaction between the D loop and the T loop/extra loop is weakened by the binding of the cognate ARS [36]. As for E coli tRNAWl, it has been indicated by a 19F <.....

Fig 4. a. The nucleotide sequence of E coli tRNA[ le arranged in the L-shaped tertiary structure. Dotted lines represent the tertiary base pairs whose imino proton resonances were assigned in the present study. Upon binding with IIeRS, imino proton resonances of base pairs (closed bars) disappeared, while those with shadowed bars were broadened and those with hatched bars were shifted. Two wobble base pairs are indicated by cross-hatched bars, because the imino proton resonances of the guanine residues in the wobble base pairs disappeared, while those of the uridine residues were not appreciably affected, b. The tertiary structure model of tRNA~le based on the crystal structure of yeast tRNA phe [33, 34]. The base pairs are marked in the same manner as in a.

1114 N M R study that the cognate ARS interacts with tRNAV~ along the entire inside of the L-shaped structure, and that a partial disruption of the interaction between T and D loops possibly occurs [37]. As discussed above, E coli IIeRS induces a global conformational change involving the dissociation of the T and D loops of tRNA[ ~e. On the other hand, as for E coli t R N A 6in and yeast tRNAAsp, it has been reported that the cognate ARSs are in contact mainly with the anticodon arm and the 5' and 3' terminal regions [10-12]. From the amino-acid sequence analysis, methionyl-, valyl-, and isoleucyl-tRNA synthetases from E coli belong to the same subgroup as the cysteinyl- and leucyl-tRNA synthetases from E coli [38]. It is proposed, therefore, that this sequence h o m o l o g y is related to a common tRNA-recognition m e c h a n i s m for these three ARSs.

Acknowledgments We thank to Dr WH McClain for discussions. This work was supported in part by a Bioscience Grant for International Joint Research Project from NEDO, Japan, a Research Grant from the International Human Frontier Science Program Organization, and Grants-in-Aid for Scientific Research on Priority Areas 01656002, 02238102, 03222102, 03242203, 04226203, 04272102, and 04272103 from the Ministry of Education, Science and Culture of Japan.

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