Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl-tRNA synthetase

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N-7 in guanosines and N-3 in cytidines by dimethyl sulfate and carbethoxylation of N-7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNATrp in solution as well as its interactions with tryptophanyl-tRNA synthetase. The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and T psi arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPhe since beef tRNATrp showed a more stable D stem and a less stable T psi stem. When alkylation by ethylnitrosourea was studied with the tRNATrp X tryptophanyl-tRNA synthetase complex we found that phosphates located at the 5' side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N-3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase X tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl-tRNA synthetase was confirmed in nuclease S1 mapping experiments.     

The effect of tRNA and tryptophanyl adenylate on limited proteolysis of beef pancreas tryptophanyl-tRNA synthetase.

Limited proteolysis of tryptophanyl-tRNA synthetase was used to detect changes in the enzyme molecule in the presence of substrates. Trypsinolysis of each of the two identical subunits occurs in succession from the N-terminus as follows: 60 leads to 51 leads to 40 leads to 24 kilodaltons. The transition 51 leads to 40 is hindered in tryptophanyl adenylate.enzyme complex. Yeast tRNATrp accelerates the first steps of hydrolysis and decelerates the transition 40 leads to 24. Once tRNATrp is added to the synthetase.adenylate complex, the protective effect of the adenylate disappears. The same effects are found also in the presence of tRNATrp oxidized with NaI04 and tRNATrp lacking the 3'-terminal adenosine. Oxidized tRNATrp (but not tRNATrp without the 3'-A) accelerates tryptophan-dependent hydrolysis of ATP catalyzed by the enzyme. A scheme is proposed for the interaction of yeast tRNATrp with beef pancreas tryptophanyl-tRNA synthetase involving the association of tRNA with a positively charged site(s) of the enzyme and the changes in the conformation of enzyme manifesting itself in unfolding of the acidic N-terminal fragment of the polypeptide chain and in the exposure of the adenylate.     

Two conformations of a crystalline human tRNA synthetase-tRNA complex: implications for protein synthesis

Aminoacylation of tRNA is the first step of protein synthesis. Here, we report the co-crystal structure of human tryptophanyl-tRNA synthetase and tRNATrp. This enzyme is reported to interact directly with elongation factor 1alpha, which carries charged tRNA to the ribosome. Crystals were generated from a 50/50% mixture of charged and uncharged tRNATrp. These crystals captured two conformations of the complex, which are nearly identical with respect to the protein and a bound tryptophan. They are distinguished by the way tRNA is bound. In one, uncharged tRNA is bound across the dimer, with anticodon and acceptor stem interacting with separate subunits. In this cross-dimer tRNA complex, the class I enzyme has a class II-like tRNA binding mode. This structure accounts for biochemical investigations of human TrpRS, including species-specific charging. In the other conformation, presumptive aminoacylated tRNA is bound only by the anticodon, the acceptor stem being free and having space to interact precisely with EF-1alpha, suggesting that the product of aminoacylation can be directly handed off to EF-1alpha for the next step of protein synthesis.     

Structural analogies between the 3' tRNA-like structure of brome mosaic virus RNA and yeast tRNATyr revealed by protection studies with yeast tyrosyl-tRNA synthetase

Contacts between the tRNA-like domain in brome mosaic virus RNA and yeast tyrosyl-tRNA synthetase have been determined by footprinting with enzymatic probes. Regions in which the synthetase caused protections indicative of direct interaction coincide with loci identified by mutational studies as being important for efficient tyrosylation [Dreher, T. W. & Hall, T. C. (1988) J. Mol. Biol. 201, 41-55]. Additional extensive contacts were found upstream of the core of the tRNA-like structure. In parallel, the contacts of yeast tRNATyr with its cognate synthetase were determined by the same methodology and comparison of protected nucleotides in the two RNAs has permitted the assignment of structural analogies between domains in the viral tRNA-like structure and tRNATyr. Amino acid acceptor stems are similarly recognized by yeast tyrosyl-tRNA synthetase in the two RNAs, indicating that the pseudoknotted fold in the viral RNA does not perturb the interaction with the synthetase. A further important analogy appears between the anticodon/D arm of the L-conformation of tRNAs and a complex branched arm of the viral tRNA-like structure. However, no apparent anticodon triplet exists in the viral RNA. These results suggest that the major determinants for tyrosylation of yeast tRNATyr lie outside the anticodon stem and loop, possibly in the amino acid acceptor stem.     

Binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase from bovine pancreas under pH conditions of maximum activity. Analysis by ultracentrifugation, fluorescence quenching and chemical modification

The binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef is examined by three approaches, under pH conditions of maximum activity (pH 8.0). (1) Analytical ultracentrifugation evidences the binding of a single mol of tRNATrp in a 2.5-10 microM concentration range. (2) tRNATrp quenches the fluorescence of the enzyme. The dependence of this fluorescence quenching on the tRNATrp concentration (0.1-4 microM) reflects also the binding of 1 mol of tRNA per mol of enzyme, with a Kd value of 0.19 +/- 0.02 microM. (3) tRNATrp protects the enzyme against derivatization by oxidized ATP. Out of the two fast-reacting lysine residues of the native enzyme, only one is prevented from reacting by tRNATrp in the 0.5-110 microM concentration range. This protection can be significantly analyzed only by assuming a one-to-one complex between the enzyme and tRNA. These results, obtained at pH 8.0 and 25 degrees C, are in contrast with the stoichiometry of 2 mol of tRNA to 1 mol of enzyme, previously observed at pH 6.0 and 4 degrees C.     

Study of the interaction of Escherichia coli methionyl-tRNA synthetase with tRNAfMet using chemical and enzymatic probes

The accessibility of nucleotides in Escherichia coli tRNAfMet to chemical and enzymatic probes in the presence and absence of methionyl-tRNA synthetase has been investigated. Dimethyl sulfate was used to probe the reactivity of cytosine and guanosine residues. The N-3 position of the wobble anticodon base, C34, was strongly protected from methylation in the tRNA-synthetase complex. A synthetase-induced conformational change in the anticodon loop was suggested by the enhanced reactivity of C32 in the presence of enzyme. Cytosine residues in the dihydrouridine loop and in the 3'-terminal CCA sequence showed little or no change in reactivity. Methylation of the N-7 position of guanosine residues G42, G52, and G70 was partially inhibited by the synthetase. Nuclease digestion of tRNAfMet with alpha-sarcin in the presence of 1-2 mM Mg2+ resulted in cleavage mainly at C71 in the acceptor stem and was strongly inhibited by synthetase. Other nuclease digestion experiments using the single strand specific nucleases RNase A and RNase T1 revealed weak protection of nucleotides in the D loop and strong protection of nucleotides in the anticodon on complex formation. The present data, together with previous structure-function studies on this system, indicate strong binding of methionyl-tRNA synthetase to the anticodon of tRNAfMet, leading to a change in the conformation of the anticodon loop and stem. We propose that this, in turn, produces more distant, and possibly relatively subtle, conformational changes in other parts of the tRNA structure that ultimately lead to proper orientation of the 3' terminus of the tRNA with respect to the active site of the enzyme.     

Role of acceptor stem conformation in tRNAVal recognition by its cognate synthetase.

Although the anticodon is the primary element in Escherichia coli tRNAValfor 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 tRNAValdemonstrates 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 tRNAAlaand 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 tRNAValverifies 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 tRNAValreduces the efficiency of aminoacylation, due largely to an increase in K m. 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 by19F 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 tRNAValby 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.     

Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E. coli: a potential application for the purification of the enzyme.

E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp. However, in the presence of Mg++, only tRNATrp can dissociate the complex whereas ATP has no effect. E. coli total tRNA or tRNAMet, at the same concentration, cannot displace the synthetase from the complex. It is suggested that the Blue-dextran binds to the synthetase through its tRNA binding domain. This hypothesis is supported by previous findings with polynucleotide phosphorylase showing that Blue-dextran Sepharose can be used in affinity chromatography to recognize a polynucleotide binding site of the protein. The selective elution by its cognate tRNA of Trp-tRNA synthetase bound to Blue-dextran Sepharose provides a rapid and efficient purification of the enzyme. Examples of other synthetases and nucleotidyl transferases are also discussed.     

Monoclonal antibodies against tryptophanyl-tRNA-synthetase

Monoclonal antibodies designated as Am1 and Am2 were prepared against purified beef pancreas tryptophanyl-tRNA synthetase (EC 6.1.1.2). Both antibodies were able to bind the native enzyme in a solid-phase assay and to precipitate enzyme activity in immune complexes. Am2 inhibited the tryptophanyl-tRNA synthetase activity in ATP-[32P]pyrophosphate exchange and in tRNATrp aminoacylation reactions; Am1 had no influence on both the enzyme activity and the inhibiting action of Am2. Only Am2, not Am1, bound elastase-modified form of the enzyme which consists of two subunits shortened by 20 000 daltons from the N-end of the molecule. These results were interpreted as an evidence for non-overlapping of Am1 and Am2 antigenic determinants along the polypeptide chains of the enzyme.     

Crystal structure of human tryptophanyl-tRNA synthetase catalytic fragment: insights into substrate recognition, tRNA binding, and angiogenesis activity

Human tryptophanyl-tRNA synthetase (hTrpRS) produces a full-length and three N terminus-truncated forms through alternative splicing and proteolysis. The shortest fragment that contains the aminoacylation catalytic fragment (T2-hTrpRS) exhibits the most potent angiostatic activity. We report here the crystal structure of T2-hTrpRS at 2.5 A resolution, which was solved using the multi-wavelength anomalous diffraction method. T2-hTrpRS shares a very low sequence homology of 22% with Bacillus stearothermophilus TrpRS (bTrpRS); however, their overall structures are strikingly similar. Structural comparison of T2-hTrpRS with bTrpRS reveals substantial structural differences in the substrate-binding pocket and at the entrance to the pocket that play important roles in substrate binding and tRNA binding. T2-hTrpRS has a wide opening to the active site and adopts a compact conformation similar to the closed conformation of bTrpRS. These results suggest that mammalian and bacterial TrpRSs might use different mechanisms to recognize the substrate. Modeling studies indicate that tRNA binds with the dimeric enzyme and interacts primarily with the connective polypeptide 1 of hTrpRS via its acceptor arm and the alpha-helical domain of hTrpRS via its anticodon loop. Our results also suggest that the angiostatic activity is likely located at the alpha-helical domain, which resembles the short chain cytokines.     

Acceptor stem and anticodon RNA hairpin helix interactions with glutamine tRNA synthetase

The class I glutamine (Gln) tRNA synthetase interacts with the anticodon and acceptor stem of glutamine tRNA. RNA hairpin helices were designed to probe acceptor stem and anticodon stem-loop contacts. A seven-base pair RNA microhelix derived from the acceptor stem of tRNA^Gln was aminoacylated by Gln tRNA synthetase. Variants of the glutamine acceptor stem microhelix implicated the discriminator base as a major identity element for glutaminylation of the RNA helix. A second RNA microhelix representing the anticodon stem-loop competitively inhibited tRNA^Gln charging. However, the anticodon stem-loop microhelix did not enhance aminoacylation of the acceptor stem microhelix. Thus, transduction of the anticodon identity signal may require covalent continuity of the tRNA chain to trigger efficient aminoacylation.     

Site selection by Xenopus laevis RNAase P

Investigation of the mechanism of cleavage site selection by Xenopus RNAase P reveals that the acceptor stem, a 7 bp helix common to all tRNA precursors, is required for cleavage. We propose that Xenopus RNAase P recognizes conserved features of the mature tRNA and that the cleavage site is selected by measuring the length of the acceptor stem. In support of this, we demonstrate that insertion of 2 bp in the acceptor stem of yeast pre-tRNA(3Leu) relocates the cleavage site 2 bases 3' to the original one. In addition, insertion of 1 bp in the acceptor stem of the end-matured yeast pre-tRNA(Phe) generates an RNAase P cleavage site: the enzyme produces a mature tRNA with the characteristic 7 bp stem and releases one 5' flanking nucleotide. Since it has previously been shown that cleavage sites of the splicing endonuclease are determined by the length of the anticodon stem, RNAase P and the splicing endonuclease apparently use different stems to determine their cutting sites.     

Transfer RNA control of the activation of isomeric tRNATrp's.

Previous studies of the homologous aminoacylations of Escherichia coli and yeast tRNATrp's terminating in 2'- and 3'-deoxyadenosine established that E. coli tryptophanyl-tRNA synthetase activates its cognate tRNA preferentially on the 2' position, while the corresponding yeast enzyme utilizes the 3' position on its homologous substrate tRNA. As this seemed to be the only change in positional specificity during evolution, the heterologous activations were investigated in an effort to determine the basis for this change. Remarkably, E. coli tRNATrp terminating in 3'-deoxyadenosine was found to be the preferred substrate for both the E. coli and yeast activating enzymes, while the same tryptophanyl-tRNA synthetase preparations both activated the isomeric yeast tRNATrp's preferentially on the 3' position. Thus, the preferred position of activation was found to be specified by the tRNA rather than the activating enzyme and, additionally, to be due to some process not reflected in initial velocity measurements. The variable utilization of individual modified aminoacyl-tRNA's as substrates in an enzyme-catalyzed deacylation process appears to provide the most likely explanation for the experimental observations.     

A mammalian tryptophanyl-tRNA synthetase shows little homology to prokaryotic synthetases but near identity with mammalian peptide chain release factor

Determination of the amino acid sequence of beef pancreas tryptophanyl-tRNA synthetase was undertaken through both cDNA and direct peptide sequencing. A full-length cDNA clone containing a 475 amino acid open reading frame was obtained. The molecular mass of the corresponding peptide chain, 53,728 Da, was in agreement with that of beef tryptophanyl-tRNA synthetase, as determined by physicochemical methods (54 kDa). Expression of this clone in Escherichia coli led to tryptophanyl-tRNA synthetase activity in cell extracts. The open reading frame included two sequences analogous to the consensus sequences, HIGH and KMSKS, found in class I aminoacyl-tRNA synthetases. The homology with prokaryotic and yeast mitochondrial tryptophanyl-tRNA synthetases was low and was limited to the regions of the consensus sequences. However, a 90% homology was observed with the recently described rabbit peptide chain release factor (eRF) [Lee et al. (1990) Proc. Natl. Acad. Sci. 87, 3508-3512]. Such a strong homology may reveal a new group of genes deriving from a common ancestor, the products of which could be involved in tRNA aminoacylation (tryptophanyl-tRNA synthetase) or translation termination (eRF).     

Molecular aspects of the inactivation of tryptophanyl transfer ribonucleic acid synthetase by N-ethylmaleimide.

The tryptic maps of tryptophanyl-tRNA synthetase from beef pancreas show that the 8 cysteinyl residues of the enzyme subunit are located, 2 by 2, on four different peptides. The kinetics of the incorporation of radioactivity from N-[ethyl-14C]ethylmaleimide into these peptides are compared in this paper with the kinetics of the changes of the catalytic properties of the enzyme occurring during alkylation. This comparison allows the identification of (a) the peptide carrying the cysteinyl residues located on the surface of the molecule, (b) the peptide carrying the deeply buried residues unmasked by the dissociation of the subunits, and (c) the peptide carrying the --SH group located in the vicinity of the binding site of tryptophan. The fourth peptide is shown to have a great sensitivity to pH with respect to the reactivity of its cysteinyl residues toward N-ethylmaleimide. The same unusual pH dependence is found for the rate of quenching of the intrinsic fluorescence of the protein during the alkylation, suggesting a strong sensitivity of the conformation of tryptophanyl-tRNA synthetase to pH in the range of 7 to 9.