Jean Gangloff

The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction.

The crystal structures of the various complexes formed by yeast aspartyl-tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation reaction. Native crystals of the binary complex tRNA-AspRS were soaked in solutions containing the two other substrates, ATP (or its analog AMPPcP) and aspartic acid. When all substrates are present in the crystal, this leads to the formation of the aspartyl-adenylate and/or the aspartyl-tRNA. A class II-specific pathway for the aminoacylation reaction is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signature sequences characteristic of class II aaRS (motifs 2 and 3) constitute the basic functional unit. The ATP molecule adopts a bent conformation, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class-invariant acidic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site-directed mutagenesis for their functional implication.

Factors determining the specificity of the tRNA aminoacylation reaction: Non-absolute specificity of tRNA-aminoacyl-tRNA synthetase recognition and particular importance of the maximal velocity

Summary It is generally believed that the specificity of tRNA aminoacylation results solely from a specific recognition between the aminoacyl-tRNA synthetase and the cognate tRNA. In fact, this specificity is not absolute: this is supported by the following observations (1) the existence of tRNA mischarging in homologous systems under usual aminoacylation conditions, (2) the existence of inhibitions produced by « non-cognatetRNA species in correct aminoacylation reactions, (3) the lack of specificity of AMP- and PPi- independent aminoacyl-tRNA synthetase catalysed deacylation of aminoacyl-tRNA species, (4) the isolation of complexes between aminoacyl-tRNA synthetases and non-cognate tRNA species. The affinities between aminoacyl-tRNA synthetases and non-cognate tRNA species, estimated by the Km measurements in mischarging reactions, have been found only diminished by 1 or 2 orders of magnitude as compared to the values found in specific systems, whereas the Vmax values for mischarging have been found diminished by 3 or 4 orders of magnitude. This suggests that tRNA aminoacylation depends more upon the maximal velocity of the reaction than upon the recognition between aminoacyl-tRNA synthetase and tRNA. Furthermore, we found that the recognition of a tRNA by an aminoacyl-tRNA synthetase does not seem to require the 3′ terminal part of the amino acid acceptor stem. As the importance of this part of the tRNA molecule during the aminoacylation process has been well established, it is possible that it is involved in determining the Vmax of the aminoacylation reaction, probably by positioning the 3′ terminal adenosine in the catalytic site of the enzyme. In conclusion, it appears that the specificity of the tRNA aminoacylation reaction proceeds through two discrimination mechanisms: the first one, measured by the Km, acts at the recognition level; the second one, which is more effective, is measured by the Vmax values. Competition phenomena have been observed between cognate and non-cognate tRNA species. They enhance the specificity of the tRNA aminoacylation, but their contribution to the specificity is low compared to that brought by Km and Vmax. Finally we found that a more rapid enzymatic deacylation of mischarged tRNA species (as compared to correctly charged ones) cannot be considered as a general mechanism for correction of misaminoacylation.

L‐Arginine recognition by yeast arginyl‐tRNA synthetase

The crystal structure of arginyl‐tRNA synthetase (ArgRS) from Saccharomyces cerevisiae, a class I aminoacyl‐tRNA synthetase (aaRS), with L‐Arginine bound to the active site has been solved at 2.75 Å resolution and refined to a crystallographic R‐factor of 19.7%. ArgRS is composed predominantly of α‐helices and can be divided into five domains, including the class I‐specific active site. The N‐terminal domain shows striking similarity to some completely unrelated proteins and defines a module which should participate in specific tRNA recognition. The C‐terminal domain, which is the putative anticodon‐binding module, displays an all‐α‐helix fold highly similar to that of Escherichia coli methionyl‐tRNA synthetase. While ArgRS requires tRNAArg for the first step of the aminoacylation reaction, the results show that its presence is not a prerequisite for L‐Arginine binding. All H‐bond‐forming capability of L‐Arginine is used by the protein for the specific recognition. The guanidinium group forms two salt bridge interactions with two acidic residues, and one H‐bond with a tyrosine residue; these three residues are strictly conserved in all ArgRS sequences. This tyrosine is also conserved in other class I aaRS active sites but plays several functional roles. The ArgRS structure allows the definition of a new framework for sequence alignments and subclass definition in class I aaRSs.

The class II aminoacyl-tRNA synthetases and their active site: Evolutionary conservation of an ATP binding site

Previous sequence analyses have suggested the existence of two distinct classes of aminoacyl-tRNA synthetase. The partition was established on the basis of exclusive sets of sequence motifs (Eriani et al. [1990] Nature 347:203–306). X-ray studies have now well defined the structural basis of the two classes: the class I enzymes share with dehydrogenases and kinases the classic nucleotide binding fold called the Rossmann fold, whereas the class II enzymes possess a different fold, not found elsewhere, built around a six-stranded antiparallel β-sheet. The two classes of synthetases catalyze the same global reaction that is the attachment of an amino acid to the tRNA, but differ as to where on the terminal adenosine of the tRNA the amino acid is placed: class I enzymes act on the 2′ hydroxyl whereas the class II enzymes prefer the 3′ hydroxyl group. The three-dimensional structure of aspartyl-tRNA synthetase from yeast, a typical class II enzyme, is described here, in relation to its function. The crucial role of the sequence motifs in substrate binding and enzyme structure is high-lighted. Overall these results underline the existence of an intimate evolutionary link between the aminoacyl-tRNA synthetases, despite their actual structural diversity.

Large scale purification and structural properties of yeast aspartyl-tRNA synthetase

A large scale purification procedure of bakers yeast aspartyl-tRNA synthetase is described which yields more than 200 mg pure protein starting from 30 Kg of wet commercial cells. The synthetase is an alpha 2 dimer of Mr = 125,000 +/- 5,000 which can be crystallized (J. Mol. Biol. 138, 1980, 129-135). The enzyme has an elongated shape with a Stokes radius of 50 A and a frictional ratio of 1.5. The synthetase has a tendency to aggregate but methods are described where this effect is overcome.

Aspartate identity of transfer RNAs

Structure/function relationships accounting for specific tRNA charging by class II aspartyl-tRNA synthetases from Saccharomyces cerevisiae, Escherichia coli and Thermus thermophilus are reviewed. Effects directly linked to tRNA features are emphasized and aspects about synthetase contribution in expression of tRNA(Asp) identity are also covered. Major identity nucleotides conferring aspartate specificity to yeast, E coli and T thermophilus tRNAs comprise G34, U35, C36, C38 and G73, a set of nucleotides conserved in tRNA(Asp) molecules of other biological origin. Aspartate specificity can be enhanced by negative discrimination preventing, eg mischarging of native yeast tRNA(Asp by yeast arginyl-tRNA synthetase. In the yeast system crystallography shows that identity nucleotides are in contact with identity amino acids located in the catalytic and anticodon binding domains of the synthetase. Specificity of RNA/protein interaction involves a conformational change of the tRNA that optimizes the H-bonding potential of the identity signals on both partners of the complex. Mutation of identity nucleotides leads to decreased aspartylation efficiencies accompanied by a loss of specific H-bonds and an altered adaptation of tRNA on the synthetase. Species-specific characteristics of aspartate systems are the number, location and nature of minor identity signals. These features and the structural variations in aspartate tRNAs and synthetases are correlated with mechanistic differences in the aminoacylation reactions catalyzed by the various aspartyl-tRNA synthetases. The reality of the aspartate identity set is verified by its functional expression in a variety of RNA frameworks. Inversely a number of identities can be expressed within a tRNA(Asp) framework. From this emerged the concept of the RNA structural frameworks underlying expression of identities which is illustrated with data obtained with engineered tRNAs. Efficient aspartylation of minihelices is explained by the primordial role of G73. From this and other considerations it is suggested that aspartate identity appeared early in the history of tRNA aminoacylation systems.

The primary structure of aspartate transfer ribonucleic acid from brewers yeast: I. Complete digestion with pancreatic ribonuclease and T1 ribonuclease

Abstract Highly purified tRNA Asp from brewers yeast prepared by countercurrent distribution followed by column chromatography has been completely digested with pancreatic ribonuclease and with T 1 ribonuclease. The separation and identification of the products have been carried out either by column chromatography followed by high voltage electrophoresis (in the case of T 1 ribonuclease digestion) or by this second technique alone (in the case of pancreatic ribonuclease digestion). Analyses indicate that this tRNA is composed of 75 nucleotide residues including 8 minor nucleotides. This tRNA contains a pUp nucleotide at the 5′ terminal end, the G-T-Ψ-C sequence common to all tRNAs of known structure and the oligonucleotide G-C-C-A at the 3′ terminal end. The primary structure of this tRNA has been determined after partial digestion with pancreatic and T 1 ribonucleases as stated in the subsequent paper.

Conditions optimales d'aminoacylation du tRNAAsp et du tRNATrp de levure

Summary For quantitative determination of tRNAAsp and tRNATrp, optimum aminoacylation conditions have been established by using purified tRNAs, 14C-labeled aminoacids and a partially purified synthetase preparation. Relevant solution parameters have been investigated including: pH, type and concentrations of buffers, incubation time and temperature, concentrations of tRNA, aminoacid, ATP, divalent and monovalent metal ions and aminoacyl-tRNA synthetase. Action of polyamines and organic solvents have been studied. The optimum pH for the aminoacylation of tRNAAsp is 7 and for tRNATrp it is 7.5. Best results are obtained with cacodylate buffer. Optimum ratio Mg++/ATP is 1.25 for tRNATrp and 1.75 for tRNAAsp. Phosphate 0.1–0.15 M has inhibiting effects. NaCl at a concentration above 0.2 M and Tris-HCl above 0.4 M are also inhibitors. The hydrolytic halflives of the two tRNA esters at the pH of optimum formation are 25 min for Asp-tRNAAsp and 16 min for Trp-tRNATrp.

Transfer RNA binding protein in the nucleus of Saccharomyces cerevisiae

A yeast nuclear protein that binds to tRNA was identified using a RNA mobility shift assay. Northwestern blotting and N‐terminal sequencing experiments indicate that this tRNA‐binding protein is identical to zuotin which has previously been shown to bind to Z‐DNA [(1992) EMBO J. 11, 3787–37961. Labeled TRNA and poly(dG‐m5dC) stabilized in the Z‐DNA form identify the same protein on a Northwestern blot. In a gel retardation assay poly(dG‐m5dC) in the Z‐form strongly diminishes the binding of tRNA to zuotin. These studies establish that zuotin is able to bind to both tRNA and Z‐DNA. Zuotin may be transiently associated with tRNA in the nucleus of yeast cells and play a role in its processing or transport to the cytoplasm.

The primary structure of aspartate transfer ribonucleic acid from brewers yeast: II. Partial digestions with pancreatic ribonuclease and T1 ribonuclease and derivation of complete sequence

Abstract Specific cleavage of tRNAAsp at the anticodon site with T1 ribonuclease enables the isolation of two halves of the molecule. Additional partial hydrolyses with pancreatic and T1 ribonucleases yield large oligonucleotide fragments for characterisation. A carbodiimide modification of tRNAAsp followed by pancreatic ribonuclease digestion was necessary for the determination of the primary structure of Half-fragment I. The information provided by these analyses and those described in the preceding paper have permitted the derivation of the total sequence of tRNAAsp.