RNA helices that recapitulate sequences of the
tRNA acceptor stem, including the 3′ 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 2′-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
3′-OH, approach from the major groove side (Arnez
& Moras, 1997). The class-specific 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.