Isoleucyl‐tRNA synthetase from baker's yeast and from Escherichia coli MRE 600

Abstract

For discrimination between isoleucine and 19 other amino acids by isoleucyl-tRNA synthetase from baker's yeast and froin Escherichia coli MRE 600, discrimination factors D have been determined from k,,, and K , values in amino-acylation of cognate tRNA"'-C-C-A. Factors D are also products of initial discrimination factors Z' and proof-reading factors II'; D = Z ' 17'. Factors 17' were calculated from AMP formation stoichiometries and factors Z ' as quotients of D and ll'; I ' = Djn'. II' is considered as a product of a pre-and post-transfer proof-reading factor (h" = n1Z12), Z ' as a product of initial discrimination factors I1 and I2 which are due to two steps of initial discrimination.With the yeast enzyme the highest accuracy is achieved in discrimination between isoleucine and valine (D = 38000); other D values in a high range (10000-20000) are observed for Gly, Ser, Thr, Leu and Met; the lowest factors D belong to Cys, Asp, Asn and Trp (300-700); the remaining amino acids are discriminated with medium

Structural Basis for Non-cognate Amino Acid Discrimination by the Valyl-tRNA Synthetase Editing Domain

“…The accuracy of protein synthesis depends on the specific recognition of amino acids and tRNAs by aminoacyl-tRNA synthetases (1,2). The aminoacylation of tRNA occurs in two steps.…”

“…Some aminoacyl-tRNA synthetases must discriminate their cognate amino acid from very similar ones. For example, isoleucyl-tRNA synthetase (IleRS) 1 should discriminate between the cognate isoleucine and the noncognate valine, which differs by only a single methylene group. Valyl-tRNA synthetase (ValRS) should discriminate the cognate valine from the noncognate, isosteric threonine, which has a quite similar shape and size as valine and a hydroxyl group in its side chain instead of a methyl group.…”

Selectivity and specificity of substrate binding in methionyl‐tRNA synthetase

“…However, the degree to which each aaRS uses the specificity enhancing steps varies considerably among the 20 naturally occurring amino acids and the type of aaRS. For example, tyrosyl t‐RNA synthetase has the highest specificity in the first binding step, whereas isoleucyl tRNA synthetase achieves maximum discrimination in the pre‐transfer proofreading step (Freist et al 1982, 1987, 1988; Freist and Sternbach 1988).…”

Crystal Structures of the CP1 Domain from Thermus thermophilus Isoleucyl-tRNA Synthetase and Its Complex with l-Valine

“…Crystals (space group P4 1 2 1 2; unit-cell parameters a ϭ b ϭ 102.7 Å, c ϭ 83.8 Å) were grown for 3 days to dimensions of ϳ0.3 ϫ 0.3 ϫ 0.3 mm 3 . To obtain cocrystals of the CP1 domain complexed with valine, reservoir solutions containing 100 mM valine were used, and the crystals thus obtained were transferred to a solution containing 55 mM Hepes-NaOH buffer (pH 7.0), 2.2 M (NH 4 ) 2 SO 4 , 5.5% 2-propanol, and 100 mM valine, 24 h before the data collection.…”

“…This reaction proceeds in two steps: the synthesis of an aminoacyladenylate, as an activated intermediate, from the amino acid and ATP, and the transfer of the aminoacyl moiety to the 3Ј-terminal of the cognate tRNA to yield the aminoacyl-tRNA (1). To maintain accurate protein biosynthesis, each aaRS must discriminate between its cognate amino acid and other similar amino acids (2,3). Some aaRSs, including the isoleucyl-, leucyl-, and valyltRNA synthetases (IleRS, LeuRS, and ValRS, respectively), have a specific editing activity that hydrolyzes the misaminoacylated tRNAs ("post-transfer editing") (4 -6).…”