Role of acceptor stem conformation in tRNAVal recognition by its cognate synthetase
Role of acceptor stem conformation in tRNA Val recognition by its cognate synthetaseMingsong Liu+, Wen-Chy Chu, Jack C.-H. Liu and Jack Horowitz*
Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50011, USA
Received September 22, 1997;Revised and Accepted November 3, 1997
ABSTRACT
Although the anticodon is the primary element in Escherichia coli tRNAVal for recognition by valyl-tRNA synthetase (ValRS), nucleotides in the acceptor stem and other parts of the tRNA modulate recognition. Study of the steady state aminoacylation kinetics of acceptor stem mutants of E.coli tRNAVal demonstrates that replacing any base pair in the acceptor helix with another Watson-Crick base pair has little effect on aminoacylation efficiency. The absence of essential recognition nucleotides in the acceptor helix was confirmed by converting E.coli tRNAAla and yeast tRNAPhe, whose acceptor stem sequences differ significantly from that of tRNAVal,to efficient valine acceptors. This transformation requires, in addition to a valine anticodon, replacement of the G:U base pair in the acceptor stem of these tRNAs. Mutational analysis of tRNAVal verifies that G:U base pairs in the acceptor helix act as negative determinants of synthetase recognition. Insertion of G:U in place of the conserved U4:A69 in tRNAVal reduces the efficiency of aminoacylation, due largely to an increase in Km. A smaller but significant decline in aminoacylation efficiency occurs when G:U is located at position 3:70; lesser effects are observed for G:U at other positions in the acceptor helix. The negative effects of G:U base pairs are strongly correlated with changes in helix structure in the vicinity of position 4:69 as monitored by 19F NMR spectroscopy of 5-fluorouracil-substituted tRNAVal. This suggests that maintaining regular A-type RNA helix geometry in the acceptor stem is important for proper recognition of tRNAVal by valyl-tRNA synthetase. 19F NMR also shows that formation of the tRNAVal-valyl-tRNA synthetase complex does not disrupt the first base pair in the acceptor stem, a result different from that reported for the tRNAGln-glutaminyl-tRNA synthetase complex.
Correct aminoacylation of tRNAs by aminoacyl-tRNA synthetases is crucial to maintaining the fidelity and efficiency of protein synthesis. Each synthetase must distinguish its cognate tRNA(s) from structurally similar non-cognate tRNAs. In vivo and in vitro methods of functional analysis have been developed (reviewed in 1 -3 ) to identify those nucleotides or structural features essential for accurate tRNA recognition and aminoacylation (positive determinants) and those that block recognition of non-cognate tRNAs (negative determinants). Only a limited number of nucleotides contribute to tRNA recognition. Their location in tRNA varies and their distribution and relative contributions to synthetase recognition differ from one tRNA to another. They may be scattered throughout the tRNA molecule as in yeast tRNAPhe (4 ), yeast tRNAAsp (5 ), Escherichia coli tRNAPhe (6 ), E.coli tRNAArg (7 ) and E.coli tRNASer (8 ), but are often localized in the first few base pairs of the acceptor stem, e.g., tRNAAla (9 ) and tRNAHis (10 ,11 ), or in the anticodon loop (E.coli tRNAMet) (12 ) or both [E.coli tRNAGln (13 ) and tRNAGly (14 )].
Major recognition elements essential for correct aminoacylation of E.coli tRNAVal by-valyl-tRNA synthetase (ValRS) are located in the anticodon (12 ,15 ,16 ), but nucleotides in the acceptor stem and in other parts of the tRNA (Liu et al., unpublished) may also contribute to synthetase recognition. Computer assisted comparison of E.coli and Salmonella typhimurium tRNA sequences(17 ) indicated that one of the distinguishing characteristics of valine-specific tRNAs is a conserved U4:A69 base pair in the acceptor stem. 19F NMR studies of the interaction of ValRS with 5-fluorouracil-substituted tRNAVal (18 ), and nuclease V1 footprinting experiments (15 , Liu et al., unpublished), have shown that valyl-tRNA synthetase either contacts the U4:A69 base pair directly or induces structural changes in that region of the acceptor stem.
To directly determine the contribution of nucleotides in the acceptor stem of E.coli tRNAVal, and in particular of the U4:A69 base pair, to recognition by ValRS, we have analyzed the steady state aminoacylation kinetics of mutant tRNAVal transcripts by purified ValRS. No positive synthetase recognition determinants were found in the acceptor stem. Substituting Watson-Crick base pairs for any base pair in the acceptor stem has little effect on aminoacylation. However, a G:U wobble base pair at any of several positions in the acceptor stem reduces catalytic efficiency. 19F NMR experiments with 5-fluorouracil-substituted tRNAVal ([FUra]tRNAVal) suggest that maintaining regular A-type RNA helix geometry in the acceptor stem is important for proper recognition of tRNAVal by ValRS. These results are supported by transferring identity elements of tRNAVal into the framework of E.coli tRNAAla and yeast tRNAPhe.
Escherichia coli tRNAVal was transcribed in vitro (19 ) from the recombinant phagemid, pVAL119-21, which contains the wild-type E.coli tRNAVal gene linked directly to an upstream T7 promoter (19 ). Methods similar to those described for tRNAVal (19 ,20 ) were used to construct a phagemid containing the gene for E.coli tRNAAla (UGC); a plasmid with the yeast tRNAPhe gene (21 ) was the gift of Dr O.C.Uhlenbeck (University of Colorado). Mutations were introduced into the cloned tRNA genes by site-directed mutagenesis (22 ) using mutagenic oligonucleotides synthesized by the Nucleic Acids Facility at Iowa State University. Mutants were selected by dideoxy DNA sequence analysis (23 ). Transcription was catalyzed by T7 RNA polymerase (24 ) in the presence of 4 mM of each nucleoside-5'-triphosphate [FUTP replaced UTP for synthesis of (FUra)tRNAVal] and 16 mM GMP, to produce tRNA with a 5'-terminal monophosphate (19 ). AMP replaced GMP in transcription of the tRNAVal variant having a 5'-terminal adenylate; yields of this transcript are low, but sufficient quantities were obtained for aminoacylation assays. The transcripts were purified by HPLC as described earlier (19 ). Nucleoside triphosphates (ATP, CTP, GTP and UTP) were products of the United States Biochemical Corporation (Cleveland, OH); 5-fluorouridine triphosphate was synthesized by Sierra Biochemicals (Tucson, AZ). Transfer RNA was quantified by spectrophotometric measurements at 260 nm, assuming a value of E0.1%260 = 24.
Initial rates of aminoacylation with E.coli ValRS, purified to homogeneity (18 ), were measured at 37°C, in 60 µl reaction mixtures containing 100 mM HEPES, pH 7.5, 15 mM MgCl2, 10 mM KCl, 7 mM ATP, 1 mM DTT, 99 µM [3H]valine (5 Ci/mmol) and 0.5-6.0 µM transfer RNA (determined by measuring valine acceptance at high ValRS concentration). Reactions were initiated by addition of 1 nM ValRS, and 10 µl samples were removed at the indicated times, spotted on Whatman 3MM paper, and processed as described by Bruce and Uhlenbeck (25 ). Results are averages of at least three experiments. The estimated error of the measurements is ±20%.
For NMR spectroscopy, tRNA samples were dissolved in a minimum volume of standard buffer (50 mM sodium cacodylate, pH 6.0, 15 mM MgCl2, 100 mM NaCl and 1 mM EDTA), and then dialyzed against two changes of the same buffer. The sample volume was then adjusted to 0.405 ml, and 10% (v/v) D2O was added as an internal lock signal. 19F NMR spectra were collected at 30°C on a Varian Unity 500 FT NMR spectrometer at 470 MHz by using 16K data points, with no relaxation delay and a pulse angle optimizing the Ernst condition (26 ). 19F chemical shifts are reported downfield from free 5-fluorouracil dissolved in standard buffer.
In many tRNAs, synthetase recognition determinants are located in the acceptor stem (1 -3 ). It has been suggested (16 ) that the conserved U4:A69 base pair found in bacterial tRNAVal and, to a lesser extent the G3:C70 base pair, serve as minor determinants for ValRS recognition. Our studies of the steady state aminoacylation kinetics of acceptor stem mutants of in vitro transcribed E.coli tRNAVal show that replacing the conserved U4:A69 with any other Watson-Crick base pair yields tRNAVal variants that are almost as active as wild-type tRNAVal (Table 1 ). The somewhat low activity of the G4:C69 mutant, relative kcat/Km = 0.31, may be due to rigidity of the acceptor stem as a result of the five consecutive G:C base pairs in this mutant tRNAVal.
Aminoacylation of E.coli tRNAAla and tRNAPhe variants with valine
tRNA
Km(µM)
kcat(sec-1)
kcat/Km
Relativekcat/Km
Wild-Type tRNAVal(UAC)
1.4
9.0
6.4
(1.0)
Escherichia coli tRNAAla
tRNAAla(UGC)G3:U70 (wild-type)
No detectable aminoacylationa
tRNAAla(UGC)G3:C70
46.7
0.49
0.01
0.0016
tRNAAla(UGC)A3:U70
-
-
0.0043
0.00067
tRNAAla(UAC)
1.1
0.31
0.51
0.079
tRNAAla(UAC)G3:C70
0.80
4.3
5.4
0.84
tRNAAla(UAC)A3:U70
0.73
5.0
6.9
1.07
Yeast tRNAPhe
tRNAPhe(GAA)G4:U69 (wild-type)
-
-
7.0 × 10-5
1.1 × 10-5
tRNAPhe(GAA)A4:U69
-
-
2.3 × 10-4
3.6 × 10-5
tRNAPhe(GAC)G4:U69
3.3
3.1
0.93
0.14
tRNAPhe(GAC)A4:U69
2.1
7.6
3.6
0.56
aLess than 0.02 pmol valine per pmol tRNA.
Substitution for other base pairs in the acceptor helix also has little effect on the kinetics of aminoacylation. Replacing G1:C72 with A1:U72 does not significantly lower the aminoacylation of tRNAVal (Table 1 ), indicating that the identity of the first base pair plays no appreciable role in tRNAVal recognition by ValRS. Furthermore, it is not essential that the first base be paired; the mutant tRNAVal with an A1*C72 mismatch is an excellent substrate for ValRS (Table 1 ). Liu et al. have reported (27 ) that G1*A72and G1*G72substitutions also have no effect on the rate of tRNAVal aminoacylation. Replacement of G2:C71 by either A2:U71 or C2:G71, and of G3:C70 with either C3:G70 or A3:U70, generates mutant tRNAs with relative kcat/Km values close to that of wild-type tRNAVal (Table 1 ). Mutant tRNAVal with Watson-Crick base pair-substitutions for A6:U67 and U7:A66, in the lower part of the acceptor helix, have moderately reduced specificity constants (Table 1 ), but remain good substrates for ValRS.
Additional evidence to support the conclusion that the acceptor stem of wild-type tRNAVal(UAC) lacks essential synthetase recognition determinants was obtained by converting E.coli tRNAAla(UGC) and yeast tRNAPhe(GAA) into efficient valine-accepting tRNAs. The acceptor stem sequences of both tRNAs differ considerably from that of tRNAVal (shaded areas in Fig. 1 b and c). Because the second and third positions of the anticodon are major synthetase recognition nucleotides of tRNAVal (12 ,15 ,16 ), we first prepared tRNAAla(UAC) andtRNAPhe(GAC), in which the alanine and phenylalanine anticodons were changed to valine anticodons.
Negative effects of G:U base pairs on recognition by ValRS were further characterized by introducing wobble base pairs at several positions in the acceptor stem of E.coli tRNAVal. The results (Table 3 ) indicate that the reduction in aminoacylation efficiency depends on the position and orientation of the G:U base pair. It is most pronounced with G:U at position 4:69. Mutants of tRNAVal with G4:U69 and U4:G69 substitutions are, respectively, 40- and 6-fold less efficient as substrates of ValRS than wild-type tRNAVal (Table 3 ). This is due primarily to significant increases in Km values. The negative influence of G:U base pairs on aminoacylation activity is also evident when the wobble base pair is positioned at 3:70 (Table 3 ), and the effect decreases as the wobble base pair is moved further from position 4:69. The G1:U72 variant of tRNAVal is as good a substrate for ValRS as wild-type tRNAVal (Table 3 ); Liu et al. (27 ) also observed no reduction in the rate of tRNAVal aminoacylation as a result of mutating C72 to U72.
Aminoacylation kinetics of E.coli tRNAVal with noncanonical base pairs in the acceptor stem
tRNA
Km (µM)
kcat (sec-1)
kcat/Km
Relative kcat/Km
Wild-Type tRNAVal
1.4
9.0
6.4
(1.0)
G1:C72 -> G1:U72
1.5
10.3
6.8
1.06
G2:C71 -> G2:U71
2.7
9.5
3.5
0.55
G3:C70 -> G3:U70
1.8
3.4
1.9
0.30
U4:A69 -> G4:U69
20.0
3.4
0.17
0.027
-> U4:G69
10.4
11.2
1.1
0.17
-> U4*C69
2.9
10.4
3.6
0.56
-> A4*G69
8.9
11.0
1.2
0.19
A6:U67 -> G6:U67
3.2
10.6
3.3
0.52
U7:A66 -> U7:G66
4.1
13.0
3.2
0.50
Base mismatches at the 4:69 position of the acceptor stem also affect aminoacylation. A purine-purine mismatch, A4*G69, decreases aminoacylation activity 5.5-fold, whereas the kcat/Km for a pyrimidine-pyrimidine mismatch, U4*C69, is closer to that of wild-type tRNAVal (Table 3 ).
To determine whether base mismatches reduce aminoacylation efficiency of tRNAVal by perturbing tRNA structure, the effect of G:U base pairs on acceptor helix geometry was probed by 19F NMR of tRNAVal labeled with fluorine by incorporation of 5-fluorouracil. 19F NMR is ideally suited for this purpose because of the resolution of 19F NMR spectra and the high sensitivity of the fluorine nucleus to changes in its environment. 5-Fluorouracil-substituted tRNAVal retains full aminoacylation activity despite replacement of all uracil residues by the base analog (19 ,28 ,29 ). There are 14 fluorouracil residues distributed throughout every stem and loop of the tRNA molecule (Fig. 1 d), and the 19F NMR spectrum of (FUra)tRNAVal shows a resolved peak for every incorporated FUra (19 ,30 ,31 ; Fig. 2 a). The spectrum has been completely assigned (32 ,33 ) and the assignments are indicated in Figure 2 a. 19F signals from fluorouracils paired with guanine resonate 4-5 p.p.m. downfield of those from fluorouracils paired with adenine (19 ). In the 19F NMR spectra of G:FU-containing (FUra)tRNAVal variants these appear in the region between 6.5 and 7.5 p.p.m. downfield of free FUra (Fig. 2 ) and are readily assigned by comparison to the spectrum of the wild-type tRNA (Fig. 2 a).
The G1:U72 mutant of tRNAVal retains full aminoacylation activity, with a relative specificity constant (kcat/Km) of 1.06 (Table 3 ). Incorporation of fluorouracil into this tRNA introduces an additional 19F probe that enables us to examine the effect of ValRS binding on the first (1:72) base pair in the acceptor stem of tRNAVal. Crystallographic studies have shown that the terminal U1:A72 base pair of tRNAGln is disrupted when glutaminyl-tRNA synthetase associates with the tRNA (34 ,35 ).
The 19F NMR spectrum of (FUra)tRNAVal(G1:U72) shows little difference from the spectrum of wild-type (FUra)tRNAVal, except for the additional resonance at 7.31 p.p.m. due to FU72 in the G1:FU72 base pair (compare Fig. 2 a and b). ValRS binding to the tRNA causes general line broadening as a result of the longer motional correlation time of the tRNA/ValRS complex (Fig. 3 ). Spectral changes induced by the enzyme are the same as those observed previously with wild-type tRNAVal (18 ): loss of intensity of 19F resonances corresponding to FU34, FU7 and FU67 with FU34 being affected most; broadening and shifting of FU12, FU4 and/or FU8 at higher ValRS/tRNA ratios; and a splitting of FU55 and FU64 (Fig. 3 ). No specific effects on peak FU72 are observed. The corresponding resonance continues to be visible and remains unshifted on addition of increasing amounts of ValRS (Fig. 3 ), indicating that the first base pair in the acceptor stem remains intact when ValRS interacts with tRNAVal. Opening of the G1:FU72 wobble base pair would shift the FU72 peak upfield to the central region of the 19F NMR spectrum, which has been assigned to resonances from unpaired 5-fluorouracils (32 ,33 ).
Although the acceptor helix is the site of identity determinants for many tRNAs, steady state aminoacylation kinetic studies with E.coli tRNAVal failed to identify any synthetase recognition nucleotides in this part of the tRNA (Table 1 ). Aminoacylation efficiency of tRNAVal is not significantly affected by Watson-Crick base pair substitutions at any position in the acceptor helix. The U4:A69 base pair, conserved in bacterial valine-specific tRNAs (17 ), can be substituted by other Watson-Crick base pairs or even by the pyrimidine-pyrimidine mismatch, U4:C69, with only relatively small decreases in the specificity constant (kcat/Km) for aminoacylation. The somewhat lower aminoacylation activity of the G4:C69 variant of tRNAVal (Table 1 ) is probably the result of increased acceptor stem rigidity due to the presence of five consecutive G:C base pairs, rather than to loss of a specific recognition element.
The absence of identity elements in the acceptor helix of tRNAVal was verified by transforming E.coli tRNAAla and yeast tRNAPhe into good substrates for ValRS. Wild-type E.coli tRNAAla and yeast tRNAPhe are very poorly aminoacylated by ValRS (Table 2 ). Converting the anticodons to a valine anticodon improves valine accepting activity. But only when the G:U base pairs in the acceptor stem are replaced with Watson-Crick base pairs do these tRNAs become good valine acceptors (Table 2 ), despite the absence of U4:A69 and other differences in acceptor stem sequence. These results support the conclusion that nucleotides in the acceptor helix of tRNAVal are not specifically recognized by ValRS, and suggest that G:U base pairs in the acceptor helix act as negative determinants to prevent proper recognition by ValRS.
Mutational analysis of tRNAVal demonstrates that introduction of G:U wobble base pairs or a purine-purine mismatch into the acceptor stem at or in the vicinity of the 4:69 base pair, at 2:71, 3:70 or 4:69, lowers the aminoacylation efficiency (kcat/Km) of the tRNA (Table 3 ). The G4:U69 mutant of tRNAVal is 40 times less efficient as a substrate for ValRs than wild-type tRNAVal, primarily because of an increase in Km (Table 3 );the aminoacylation efficiency of the U4:G69 mutant is also reduced. A A4:G69, purine-purine mismatch, decreases aminoacylation activity significantly (Table 3 ), whereas a U4:C69, pyrimidine-pyrimidine mismatch, has a smaller effect on activity (Table 3 ).
It seems likely that G:U wobble and mismatched base pairs inhibit aminoacylation of tRNAVal by distorting the conformation of the acceptor helix. The geometry of a G:U base pair, compared to a standard Watson-Crick base pair, involves displacement of the guanine toward the minor groove. This influences stacking interactions with adjacent base pairs (36 ,37 ) in a sequence-dependent manner (38 ). Crystallographic (36 ,39 ,40 ) and high-resolution NMR (41 ,42 ) investigations of tRNAs and short RNA duplexes have shown that G:U base pairs induce local variations in helix geometry at and around the mismatch. Such changes in helical structure are reflected in distinct chemical shift changes in the 18F NMR spectra of G:U-substituted (FUra)tRNAVal (Fig. 2 ). Introduction of G:U base pairs into the acceptor stem results in shifts of resonances assigned to FUra residues in the acceptor helix. The most prominent spectral change is the downfield shift of the signal from FU4 when G:FU substitutions are made at 2:71, 3:70 and 4:69 (Fig. 2 ; Table 4 ). The direct relationship between the magnitude of this shift and the decrease in amino acid accepting activity of the tRNA (Table 4 ) strongly suggests a correlation between aminoacylation efficiency and acceptor helix conformation in the vicinity of the 4:69 base pair. These results lead us to conclude that ValRS does not specifically recognize the U4:A69 base pair and that an unperturbed A-form helical structure in the middle of the acceptor stem is required for productive interaction of the enzyme with tRNAVal.
Solution of the X-raystructure of the complex of tRNAGln with glutaminyl-tRNA synthetase, a class I synthetase like ValRS, shows that the 3' end of the tRNA loops back toward the anticodon, disrupting the 1:72 base pair in the acceptor stem (34 ,35 ). Availability of the G1:FU72 variant of (FUra)tRNAVal, which is fully active in aminoacylation (Table 3 ), permitted us to use 19F NMR to investigate the effect of ValRS binding on the integrity of the acceptor helix of tRNAVal. 19F spectra of this tRNAVal variant show that the 1:72 base pair remains intact as increasing amounts of ValRS bind the tRNA (Fig. 3 ). Evidently, synthetase-induced disruption of the first base pair in the acceptor stem is not a general characteristic of class I synthetases.
Support for this investigation was provided by grant MCB 95-13932 from the National Science Foundation. We thank Vahid Feiz for construction of the plasmid containing the gene for E.coli tRNAAla, Olke Uhlenbeck (University of Colorado, Boulder) for the yeasttRNAPhe-containing plasmid, and Shimin Li and Diane Shogren for technical support. This is Journal Paper No. J-17573 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA. Project No. 3131.
38 Moras,D., Dumas,P. and Westhof,E. (1986) In van Knippenberg,P.H. and Hilbers,C.W. (eds), Structure and Dynamics of RNA. Plenum Press, New York, NY, pp. 113-124.
42 Ramos,A. and Varani,G. (1997) Nucleic Acids Res.,25, 2083-2090.MEDLINE Abstract
*To whom correspondence should be addressed. Tel: +1 515 294 8344; Fax: +1 515 294 0453; Email: jhoro@iastate.edu +Present address: Cardiovascular Research Institute, University of California-San Francisco, San Francisco, CA 94143, USA
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