acid bound to a class I tRNA synthetase
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For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended β1–β2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNA Trp , which could be partially rescued by adding tRNA Trp , even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode.
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Complexes between tRNA Phe (yeast), tRNA Ser (yeast) and tRNA Tyr ( Escherichia coli ) and their cognate aminoacyl‐tRNA synthetases have been studied by sedimentation velocity runs in an analytical ultracentrifuge. The amount of complex formation was determined by the absorption and the sedimentation coefficients of the fast‐moving boundary in the presence of excess tRNA or excess synthetase respectively. The same method has been applied to unspecific combinations of tRNAs and synthetases. Inactive material of tRNA or synthetase does not influence the results. Two moles of tRNA Phe can be bound to one mole of phenylalanyl‐tRNA synthetase with a binding constant ≫ 10 6 M −1 . The binding constants for both tRNAs are very similar; the binding sites are independent of each other. Omission of Mg 2+ does not prevent binding. Two moles of tRNA Ser can be bound to one mole of Seryl‐tRNA synthetase; the binding of the first and second tRNA is non‐equivalent. K 1 ≫ 10 6 M −1 , K 2 is determined to be 1.3×10 5 M −1 at pH 7.2. Omission of Mg 2+ prevents complex formation. Tyrosyl‐tRNA synthetase behaves very similarly to seryl‐tRNA synthetase. The binding constant for the weakly bound tRNA is 2.3×10 5 M −1 at pH 7.2, and 2.5×10 6 M −1 at pH 6.0. No complexes are observed in the absence of Mg 2+ . Unspecific binding was only obtained with phenylalanyl‐tRNA synthetase. It binds tRNA Ser (yeast), tRNA Ala (yeast) and tRNA Tyr ( E. coli ) with a binding constant about 100 times lower compared to its cognate tRNA. The binding data are discussed with respect to the tertiary structure of the tRNAs, the subunit structure of the synthetases and the possible physical basis for the non‐equivalence of binding sites.
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