Nucleic Acids Research, Vol 27, Issue 24 4823-4829, Copyright © 1999 by Oxford University Press
T Stello, M Hong and K Musier-Forsyth
In this work, we probe the role of the anticodon in tRNA recognition by
human lysyl-tRNA synthetase (hLysRS). Large decreases in aminoacylation
efficiency are observed upon mutagenesis of anticodon positions U35 and U36
of human tRNA(Lys,3). A minihelix derived from the acceptor-TPsiC stem-loop
domain of human tRNA(Lys,3)was not specifically aminoacylated by the human
enzyme. The presence of an anticodon-derived stem-loop failed to stimulate
aminoacylation of the minihelix. Thus, covalent continuity between the
acceptor stem and anticodon domains appears to be an important requirement
for efficient charging by hLysRS. To further examine the mechanism of
communication between the critical anticodon recognition elements and the
catalytic site, a two piece semi- synthetic tRNA(Lys, 3)construct was used.
The wild-type semi-synthetic tRNA contained a break in the phosphodiester
backbone in the D loop and was an efficient substrate for hLysRS. In
contrast, a truncated variant that lacked nucleotides 8-17 in the D
stem-loop displayedseverely reduced catalytic efficiency. The elimination
of key tRNA tertiary structural elements has little effect on
anticodon-dependent substrate binding but severely impacts formation of the
proper transition state for catalysis. Taken together, our studies provide
new insights into human tRNA structural requirements for effective
transmission of the anticodon recognition signal to the distal acceptor
stem domain.
ARTICLES
Efficient aminoacylation of tRNA(Lys,3) by human lysyl-tRNA synthetase is dependent on covalent continuity between the acceptor stem and the anticodon domain
Department of Chemistry, University of Minnesota, 207 Pleasant Street S.E., Minneapolis, MN 55455, USA.
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