Aminoacyl-tRNA synthetases from yeast: generality of chemical proofreading in the prevention of misaminoacylation of tRNA

Gl, Igloi; F, von der Haar; Cramer, Friedrich

doi:10.1021/bi00610a006

Cited by 61 publications

(56 citation statements)

References 52 publications

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“…The existence of a second binding site for the correction remains to be demonstrated. Proofreading has been well studied for some enzymes of class I which aminoacylate hydrophobic amino acids--ValRS and IleRS--where it plays a pivotal role in accuracy of the aminoacylation reaction (Igloi et al 1978;Freist et al 1987). For other enzymes like ArgRS and TyrRS such reaction seems to be marginal, the pretransfer proofreading reaction being the main correction step (Freist et al 1989;Freist and Sternbach 1988).…”

Section: Functional and Evolutionary Considerationsmentioning

confidence: 99%

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

Eriani¹,

Cavarelli²,

Martin³

et al. 1995

J Mol Evol

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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 beta-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.

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Section: Functional and Evolutionary Considerationsmentioning

confidence: 99%

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

Eriani¹,

Cavarelli²,

Martin³

et al. 1995

J Mol Evol

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“…The CP1 domain of both of these enzymes has been shown to be responsible for editing threonine in the case of ValRS and isoleucine and methionine in the case of LeuRS (8, 9, 14 -16). Similar to IleRS, the CP1 domains of ValRS and LeuRS are highly conserved (9,15), and editing activity is present in a range of species examined thus far (9,14,15,(17)(18)(19)(20)(21)(22).…”

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confidence: 99%

Species-specific Differences in Amino Acid Editing by Class II Prolyl-tRNA Synthetase

Beuning

Musier‐Forsyth

2001

Journal of Biological Chemistry

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Aminoacyl-tRNA synthetases are a family of enzymes responsible for ensuring the accuracy of the genetic code by specifically attaching a particular amino acid to their cognate tRNA substrates. Through primary sequence alignments, prolyl-tRNA synthetases (ProRSs) have been divided into two phylogenetically divergent groups. We have been interested in understanding whether the unusual evolutionary pattern of ProRSs corresponds to functional differences as well. Previously, we showed that some features of tRNA recognition and aminoacylation are indeed groupspecific. Here, we examine the species-specific differences in another enzymatic activity, namely amino acid editing. Proofreading or editing provides a mechanism by which incorrectly activated amino acids are hydrolyzed and thus prevented from misincorporation into proteins. "Prokaryotic-like" Escherichia coli ProRS has recently been shown to be capable of misactivating alanine and possesses both pretransfer and post-transfer hydrolytic editing activity against this noncognate amino acid. We now find that two ProRSs belonging to the "eukaryotic-like" group exhibit differences in their hydrolytic editing activity. Whereas ProRS from Methanococcus jannaschii is similar to E. coli in its ability to hydrolyze misactivated alanine via both pretransfer and post-transfer editing pathways, human ProRS lacks these activities. These results have implications for the selection or design of antibiotics that specifically target the editing active site of the prokaryotic-like group of ProRSs.

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“…The esterification of ['4C]leucine to unfractionated tRNA was measured as described [15,181. The standard assay was performed at 37°C in 0.1 ml solution containing 150 mM Tris/HCl buffer pH 7.65, 150 mM KC1, 10 mM MgS04, 2 mM ATP, 5 mM 2-mercaptoethanol, 1 mg unfractionated tRNA and 0.03 mM labeled amino acid.…”

Section: Aminoacylationmentioning

confidence: 99%

Biochemical comparison of the Neurospora crassa wild type and the temperature‐sensitive and leucine‐auxotroph mutant leu‐5

Kunugi

Uehara-Kunugi

Haar

et al. 1986

European Journal of Biochemistry

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The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 "C) and mutant (grown at 28 "C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28°C and transferred then to 37°C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37°C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish any difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 "C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of-1 : 600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme. The mutant leu-5 of Neurospora crassa, which is temperature sensitive and leucine-auxotroph, has been reported earlier to have a mutation in the leucyl-tRNA synthetase gene, providing a cytoplasmic leucyl-tRNA synthetase with an increased K, for leucine, a protein biosynthesis with one or some inaccurate steps and very little mitochondrial leucyl-tRNA synthetase [ 11. These phenomena have been tentatively explained by Gross et al. in a proposal regarding the structure and function of the corresponding genes [2-41. Enzyme. Leucyl-tRNA synthetase (EC 6.1.1.4). However, the molecular biology of the mutation has not been established at the DNA level nor at the protein level, and this will be of interest regarding the structure and function of aminoacyl-tRNA synthetases. Moreover a leucyl-tRNA synthetase with reduced function may establish th...

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Aminoacyl-tRNA synthetases from yeast: generality of chemical proofreading in the prevention of misaminoacylation of tRNA

Cited by 61 publications

References 52 publications

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

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

Species-specific Differences in Amino Acid Editing by Class II Prolyl-tRNA Synthetase

Biochemical comparison of the Neurospora crassa wild type and the temperature‐sensitive and leucine‐auxotroph mutant leu‐5

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