Fidelity in the aminoacylation of tRNAVal with hydroxy analogs of valine, leucine, and isoleucine by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli

Abstract

Several analogues of valine, leucine, and isoleucine carrying hydroxyl groups in the gamma- or delta-position have been tested in the aminoacylation of tRNA by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli. Results of the ATP/PPi exchange and of the aminoacylation reactions indicate that the amino acid analogues not only can form the aminoacyl adenylate intermediate but are also transferred to tRNA. However, the fact that the reaction consumes an excess of ATP indicates that the misactivated amino acid analogue is hydrolytically removed. Thus, valyl-tRNA synthetase from S. cerevisiae shows a high fidelity in forming valyl-tRNA. Although the much bulkier amino acid analogues allo- and iso-gamma-hydroxyvaline and allo- and iso-gamma-hydroxyisoleucine are initially charged to tRNA, the misaminoacylated tRNA(Val) is enzymatically deacylated. This cleavage reaction is mediated by the hydroxyl groups of the amino acid analogues which are converted into the corresponding lactones.

Homoserine Toxicity in Saccharomyces cerevisiae and Candida albicans Homoserine Kinase ( thr1 Δ) Mutants

“…Similarly, the lack of suppression of homoserine toxicity by expression of the E. coli threonyl-tRNA synthetase, which does not aminoacylate homoserine (30), argues against a role for homoserine incorporation into proteins via only Ths1p-mediated aminoacylation. However, since translation is required for homoserine toxicity, homoserine may be misacylated by multiple aminoacyl tRNA synthetases, as is the case for homocysteine (12,13,(41)(42)(43)(44). Various homoserinemediated deleterious phenotypes observed for thr1⌬ and thr4⌬ mutants would be consistent with homoserine incorporation into proteins, creating aberrant proteins that are sensitive to various stresses, analogous to effects mediated by the incorporation into protein of toxic amino acid analogs (93,96).…”

Omnipresent Maxwell's demons orchestrate information management in living cells

“…For example, Cramer and co-workers illustrated the problem by comparing the input of valine and isoleucine analogues in the corresponding tRNA synthetases. They found that several energy-consuming processes were implemented during the apparently simple process of ligation of an amino acid to its cognate tRNA (Englisch-Peters et al, 1990). The conclusion was that standard recognition was far too low to allow accurate discrimination.…”

Editing of errors in selection of amino acids for protein synthesis.

“…Isoleucyl-tRNA synthetase exhibits a weak deacylase activity toward Ile-tRNAI1e (k = 0.8 min-1 [124]) and a strong deacylase activity toward Val-tRNAI'le (k = 10 s-1 [26,31]), but Val-tRNAIle does not form during editing of valine by isoleucyl-tRNA synthetase (31). Valyl-tRNA synthetase possesses a weak deacylase activity toward Val-tRNAVal (k = 0.02 s-1 [ Table 1]) and edits misactivated threonine via deacylation of Thr-tRNAVal (k = 40 s-1 [37]), yet it has been reported to form stable homocysteinyl-tRNAVal (28). Leucyl-tRNA synthetase is known to possess an efficient deacylase activity toward Leu-tRNAJU (k = 0.08 s-1 [71]), but nevertheless it forms mischarged homocysteinyl-tRNAIeu which does not seem to be deacylated by the enzyme (27).…”

“…This suggested that a 2'-OH is an acceptor site for amino acids and that a 3'-OH is required for deacylation. The requirement for the 3'-OH was explained by proposing that it activates a water molecule that participates in hydrolysis of the adjacent bond of aminoacyl-tRNA (chemical proofreading [28,67,68,145]). Alternatively, the 3'-OH may be required so that the amino acid can migrate onto it and become accessible to the hydrolytic site (hydrolytic editing [41]).…”

“…Although several aminoacyl-tRNA synthetases (MetRS, IleRS, ValRS, and LeuRS) edit homocysteine by converting it into the thiolactone in vitro, only one synthetase, i.e., methionyl-tRNA synthetase, is involved in the thiolactone synthesis in living cells. In preventing errors, it is important that IleRS, ValRS, and LeuRS do not interact with homocysteine in vivo, since at least LeuRS and ValRS form homocysteinyl-tRNAIeu (27) and homocysteinyl-tRNAVal (28), respectively, in vitro.…”