Recognition of functional groups in an RNA helix by a class I tRNA synthetase.

RNA helices that recapitulate sequences of the tRNA acceptor stem, including the 39 NCCA nucleotides, can be substrates for aminoacyl–tRNA synthetases (Frugier et al+, 1994; Hamann & Hou, 1995; Martinis & Schimmel, 1995; Quinn et al+, 1995)+ Although the catalytic efficiency of aminoacylation of RNA helices is reduced from that of the full-length parent tRNA, the specificity is maintained+ The specific aminoacylation lies in the ability of aminoacyl–tRNA synthetases to recognize functional groups within the RNA helices+ Analysis of tRNA–synthetase structures has suggested a general principle (Rould et al+, 1989; Ruff et al+, 1991; Arnez & Moras, 1997)+ The class I synthetases, which attach an amino acid initially to the 29-OH of the terminal ribose, approach the acceptor and NCCA end from the minor groove side+ The class II synthetases, which attach an amino acid to the terminal 39-OH, approach from the major groove side (Arnez & Moras, 1997)+ The classspecific approach leads to tRNA–synthetase complexes that are near mirror images of each other and provides a structural rationale for the stereochemistries of aminoacylation+ We report here the identification of a functional group in the acceptor end of Escherichia coli tRNACys that is important for the class I cysteine–tRNA synthetase+ This functional group makes one of the largest energetic contributions to aminoacylation+ However, it is located on the major groove side of the acceptor stem+ Kinetic analysis of the contribution of this functional group to aminoacylation suggests new features that are not anticipated from the class-specific approach of synthetases+ The acceptor stem of E. coli tRNACys (Fig+ 1A) is a substrate for aminoacylation (Hamann & Hou, 1995)+ For example, an RNA microhelix that contains the acceptor stem, the UCCA end, and the UUCG tetra-loop (Fig+ 1B) is specifically aminoacylated with cysteine+An RNA minihelix (not shown) that extends the microhelix by including the TCC stem is also specifically aminoacylated with cysteine, with a catalytic efficiency (kcat/Km) of aminoacylation similar to that of the microhelix+ In these RNA helices, the determinant for aminoacylation is U73 (Hamann & Hou, 1995)+ Substitution of U73 with A73,C73, or G73 completely eliminates aminoacylation+ Further, transfer of U73 to RNA helices of a different specificity confers on the latter the ability to accept cysteine+ For example, introduction of U73 to the RNA helix of tRNAAla enables aminoacylation with cysteine, even though the major determinant for aminoacylation with alanine (G3:U73) is still present in the acceptor stem+ In the transplanted helix, the kcat/Km value of aminoacylation with cysteine is virtually identical to that of the helix of tRNACys, whereas kcat/Km of aminoacylation with alanine is reduced by 30-fold (Hamann & Hou, 1995)+ The dominant role of U73 in aminoacylation with cysteine is also observed in the full-length tRNACys+ In E. coli tRNACys, U73 is the most important nucleotide for aminoacylation, and it accounts for 7+1 kcal/mol of the free energy change of activation (Komatsoulis & Abelson, 1993; Hou, 1997)+ The significance of U73 in E. coli tRNACys is followed by the GCA anticodon (4+3 kcal/mol) and a G15:G48 tertiary base pair (2+8 kcal/mol) (Komatsoulis & Abelson, 1993; Lipman & Hou, 1998)+ U73 is conserved in all cysteine-specific tRNAs and its role in aminoacylation appears conserved in evolution+ Studies of the human, yeast, and several microbial cysteine tRNAs have confirmed the significance of U73 (Lipman & Hou, 1998)+ The prominence of U73 Reprint requests to: Ya-Ming Hou, Department of Biochemistry and Molecular Pharmacology,Thomas Jefferson University, 233 South 10th Street, BLSB 220, Philadelphia, Pennsylvania 19107, USA; e-mail: Ya-Ming+Hou@mail+tju+edu+ RNA (2000), 6:922–927+ Cambridge University Press+ Printed in the USA+ Copyright © 2000 RNA Society+