L-Arginine recognition by yeast arginyl-tRNA synthetase (original) (raw)
Related papers
tRNA aminoacylation by arginyl-tRNA synthetase: induced conformations during substrates binding
The EMBO Journal, 2000
The 2.2 A Ê crystal structure of a ternary complex formed by yeast arginyl-tRNA synthetase and its cognate tRNA Arg in the presence of the L-arginine substrate highlights new atomic features used for speci®c substrate recognition. This ®rst example of an active complex formed by a class Ia aminoacyl-tRNA synthetase and its natural cognate tRNA illustrates additional strategies used for speci®c tRNA selection. The enzyme speci®cally recognizes the D-loop and the anticodon of the tRNA, and the mutually induced ®t produces a conformation of the anticodon loop never seen before. Moreover, the anticodon binding triggers conformational changes in the catalytic center of the protein. The comparison with the 2.9 A Ê structure of a binary complex formed by yeast arginyl-tRNA synthetase and tRNA Arg reveals that L-arginine binding controls the correct positioning of the CCA end of the tRNA Arg. Important structural changes induced by substrate binding are observed in the enzyme. Several key residues of the active site play multiple roles in the catalytic pathway and thus highlight the structural dynamics of the aminoacylation reaction.
Journal of Molecular Biology, 2000
The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized. A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels. A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line. The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5. The corresponding mutant enzymes were expressed in yeast and puri®ed. The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP i exchange and tRNA aminoacylation reactions. This mutation is located underneath the¯oor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the a-helix bundle C-terminal domain distinctive of class 1a synthetases. For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates. However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo. In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic ef®ciency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme. In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the¯oor of the active site.
Amino Acids, 2006
Amino acids are building blocks of proteins, while aminoacyl-tRNA synthetases (aaRSs) catalyze the first reaction in such building: the biosynthesis of proteins. The E. coli arginyl-tRNA synthetase (ArgRS) has been crystallized in complex form with tRNA Arg (B. stearothermophilus), at pH 5.6 using ammonium sulfate as a precipitating agent. Two crystal forms have been identified based on unit cell dimension. The complete data sets from both crystal forms have been collected with a primitive hexagonal space group. A data set of Form II crystals at 3.2 Å and 94% completeness has been obtained, with unit cell parameters a ¼ b ¼ 98.0 Å , c ¼ 463.2 Å , and a ¼ b ¼ 90 , g ¼ 120 , being different from a ¼ b ¼ 110.8 Å , c ¼ 377.8 Å for form I. The structure determination will demonstrate the interaction of these two macromolecules to understand the special mechanism of ArgRS that requires the presence of tRNA for amino acid activation. Such complex structure also provides a wide opening for inhibitor search using bioinformatics.
Nucleic Acids Research, 1997
Gene cloning, overproduction and an efficient purification protocol of yeast arginyl-tRNA synthetase (ArgRS) as well as the interaction patterns of this protein with cognate tRNA Arg and non-cognate tRNA Asp are described. This work was motivated by the fact that the in vitro transcript of tRNA Asp is of dual aminoacylation specificity and is not only aspartylated but also efficiently arginylated. The crystal structure of the complex between class II aspartyl-tRNA synthetase (AspRS) and tRNA Asp , as well as early biochemical data, have shown that tRNA Asp is recognized by its variable region side. Here we show by footprinting with enzymatic and chemical probes that transcribed tRNA-Asp is contacted by class I ArgRS along the opposite D arm side, as is homologous tRNA Arg , but with idiosyncratic interaction patterns. Besides protection, footprints also show enhanced accessibility of the tRNAs to the structural probes, indicative of conformational changes in the complexed tRNAs. These different patterns are interpreted in relation to the alternative arginine identity sets found in the anticodon loops of tRNA Arg and tRNA Asp . The mirror image alternative interaction patterns of unmodified tRNA Asp with either class I ArgRS or class II AspRS, accounting for the dual identity of this tRNA, are discussed in relation to the class defining features of the synthetases. This study indicates that complex formation between unmodified tRNA Asp and either ArgRS and AspRS is solely governed by the proteins.
The EMBO Journal, 2000
Cytoplasmic aspartyl-tRNA synthetase (AspRS) from Saccharomyces cerevisiae is a homodimer of 64 kDa subunits. Previous studies have emphasized the high sensitivity of the N-terminal region to proteolytic cleavage, leading to truncated species that have lost the ®rst 20±70 residues but that retain enzymatic activity and dimeric structure. In this work, we demonstrate that the N-terminal extension in yeast AspRS participates in tRNA binding and we generalize this ®nding to eukaryotic class IIb aminoacyl-tRNA synthetases. By gel retardation studies and footprinting experiments on yeast tRNA Asp , we show that the extension, connected to the anticodon-binding module of the synthetase, contacts tRNA on the minor groove side of its anticodon stem. Sequence comparison of eukaryotic class IIb synthetases identi®es a lysine-rich 11 residue sequence ( 29 LSKKALKKLQK 39 in yeast AspRS with the consensus xSKxxLKKxxK in class IIb synthetases) that is important for this binding. Direct proof of the role of this sequence comes from a mutagenesis analysis and from binding studies using the isolated peptide.
Evidence that arginyl-adenylate is not an intermediate in the arginyl-tRNA synthetase reaction
Archives of Biochemistry and Biophysics, 1975
Arginyl-tRNA synthetase has a reaction mechanism not typical of most aminoacyl-tRNA synthetases. It does not catalyze an amino acid-dependent ATP-PP, exchange in the absence of tRNA as do most enzymes of this class. In order to clarify the reaction mechanism by performing experiments with substrate levels of enzyme, we have modified the previous purification procedure. By the method presented,.homogeneous enzyme can be prepared in approximately 10% yield. Pulse-labeling experiments indicate that no enzyme-bound arginyl-adenylate is formed in the absence of tRNA. Equilibrium experiments show that no arginyl-adenylate accumulates either in the presence or absence of tRNAarg. Two mechanisms compatible with these data are suggested.
An RNA Binding Site in a tRNA Synthetase with a Reduced Set of Amino Acids
Biochemistry, 1994
A 30 amino acid helix-loop of known structure on the surface of the C-terminal domain of the class I Escherichia coli methionine tRNA synthetase is essential for methionine tRNA anticodon discrimination. Replacing this 30 amino acid peptide with a previously described sequence containing residues from the wild-type protein imbedded in a sequence matrix of mostly alanines and serines, we used a combinatorial mutagenesis and selection strategy to define residual wild-type residues that are not replaceable with alanine or serine, because they are needed for specific recognition of methionine tRNA. Four were identified, of which three have functional side chains (Asn, Arg, Lys). These four and a fifth (Trp) that was previously identified are located at the end of the helix and within the loop, lie on the same side of the structure, and span a distance of about 20 A. We conclude that, within the alanine, serine sequence matrix, only a few non-alanine, non-serine residues in the specificity-determining part of the structure are essential.
Journal of Molecular Biology, 2000
Aminoacyl-tRNA synthetases catalyze the speci®c charging of amino acid residues on tRNAs. Accurate recognition of a tRNA by its synthetase is achieved through sequence and structural signalling. It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound synthetases. To address this issue the crystal structure of the dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNA Asp . The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and re®ne the structure of free AspRS at 2.3 A Ê resolution. For the ®rst time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex. Overall, the synthetase is less affected by the association than the tRNA, although signi®cant local changes occur. They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit. The most dramatic differences are observed in two evolutionary conserved loops. Both are in the neighborhood of the catalytic site and are of importance for ligand binding. The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the synthetase anticodon binding module.