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Aminoacyl-tRNA synthetases attach amino acids to the 3′
termini of cognate tRNAs to establish the specificity of protein
synthesis. A recent Asilomar conference (California, January
13–18, 2002) discussed new research into the
structure–function relationship of these crucial enzymes,
as well as a multitude of novel functions, including participation
in amino acid biosynthesis, cell cycle control, RNA splicing,
and export of tRNAs from nucleus to cytoplasm in eukaryotic
cells. Together with the discovery of their role in the cellular
synthesis of proteins to incorporate selenocysteine and
pyrrolysine, these diverse functions of aminoacyl-tRNA synthetases
underscore the flexibility and adaptability of these ancient
enzymes and stimulate the development of new concepts and methods
for expanding the genetic code.
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.
The tRNA 3′ end contains the conserved CCA
sequence at the 74–76 positions. The CCA sequence
is synthesized and maintained by the CCA-adding enzymes.
The specificity of the Escherichia coli enzyme
at each of the 74–76 positions was investigated using
synthetic minihelix substrates that contain permuted 3′
ends. Results here indicate that the enzyme has the ability
to synthesize unusual 3′ ends. When incubated with
CTP alone, the enzyme catalyzed the addition of C74, C75,
C76, and multiple Cs. Although the addition of C74 and
C75 was as expected, that of C76 and multiple Cs was not.
In particular, the addition of C76 generated CCC, which
would have conflicted with the biological role of the enzyme.
However, the presence of ATP prevented the synthesis of
CCC and completely switched the specificity to CCA. The
presence of ATP also had an inhibitory effect on the synthesis
of multiple Cs. Thus, the E. coli CCA enzyme can
be a poly(C) polymerase but its synthesis of poly(C) is
regulated by the presence of ATP. These features led to
a model of CCA synthesis that is independent of a nucleic
acid template. The synthesis of poly(C) by the CCA-adding
enzyme is reminiscent of that of poly(A) by poly(A) polymerase
and it provides a functional rationale for the close sequence
relationship between these two enzymes in the family of
The CCA end is common to all tRNAs as the universal site for amino acid attachment. It is also conserved in the 3′-terminal tRNA-like structure of viral genomes that can be aminoacylated by an aminoacyl-tRNA synthetase (Florentz & Giegé, 1995). During aminoacylation, the CCA end enters the catalytic center of an aminoacyl-tRNA synthetase and provides the site for chemistry to take place. The CCA end is also widely used in replication of retroviruses, the bacterial single-stranded RNA viruses, and duplex DNA plasmids of fungal mitochondria. During replication, the CCA end interacts with the template-specificity domain of reverse transcriptase or replicase and provides the initiation site for primer binding and extension (Maizels & Weiner, 1994). The importance of the CCA end in translation and in replication suggests that its conformation will play a role in these two fundamental processes.
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