Nucleic Acids Research, Vol 25, Issue 24 4883-4890, Copyright © 1997 by Oxford University Press
M Liu, WC Chu, JCH Liu and J Horowitz
Although the anticodon is the primary element in Escherichia coli
tRNAValfor recognition by valyl-tRNA synthetase (ValRS), nucleotides in the
acceptor stem and other parts of the tRNA modulate recognition. Study of
the steady state aminoacylation kinetics of acceptor stem mutants of E.coli
tRNAValdemonstrates that replacing any base pair in the acceptor helix with
another Watson-Crick base pair has little effect on aminoacylation
efficiency. The absence of essential recognition nucleotides in the
acceptor helix was confirmed by converting E.coli tRNAAlaand yeast tRNAPhe,
whose acceptor stem sequences differ significantly from that of tRNAVal, to
efficient valine acceptors. This transformation requires, in addition to a
valine anticodon, replacement of the G:U base pair in the acceptor stem of
these tRNAs. Mutational analysis of tRNAValverifies that G:U base pairs in
the acceptor helix act as negative determinants of synthetase recognition.
Insertion of G:U in place of the conserved U4:A69 in tRNAValreduces the
efficiency of aminoacylation, due largely to an increase in K m. A smaller
but significant decline in aminoacylation efficiency occurs when G:U is
located at position 3:70; lesser effects are observed for G:U at other
positions in the acceptor helix. The negative effects of G:U base pairs are
strongly correlated with changes in helix structure in the vicinity of
position 4:69 as monitored by19F NMR spectroscopy of
5-fluorouracil-substituted tRNAVal. This suggests that maintaining regular
A-type RNA helix geometry in the acceptor stem is important for proper
recognition of tRNAValby valyl-tRNA synthetase.19F NMR also shows that
formation of the tRNAVal-valyl-tRNA synthetase complex does not disrupt the
first base pair in the acceptor stem, a result different from that reported
for the tRNAGln-glutaminyl- tRNA synthetase complex.
ARTICLES
Role of acceptor stem conformation in tRNAVal recognition by its cognate synthetase
Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50011, USA.
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