Nucleic Acids Research, Vol 25, Issue 22 4551-4556, Copyright © 1997 by Oxford University Press
AU Metzger, M Heckl, D Willbold, K Breitschopf, UL RajBhandary, P Rosch and HJ Gross
Correct recognition of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases
(aaRS) is crucial to the maintenance of translational fidelity. The
discriminator base A73 in human tRNALeuis critical for its specific
recognition by the aaRS. Exchanging A73 for G abolishes leucine acceptance
and converts it into a serine acceptor in vitro . Two RNA microhelices of
24 nt length that correspond to the tRNALeuacceptor stem and differ only in
the discriminator base were synthesized: a wild-type tRNALeumicrohelix,
where nt 21 corresponds to the discriminator base position 73, and an A21G
mutant microhelix. To investigate whether different identities of both
tRNAs are caused by conformational differences, NMR and UV melting
experiments were performed on both microhelices. Two-dimentional NOESY
spectra showed both microhelices to exhibit the same overall conformation
at their 3'- CCA ends. Thermodynamic analysis and melting behaviour of the
base- paired imino protons observed by NMR spectroscopy suggest that the
A21G (A73G in tRNA) exchange results in a decrease of melting transition
cooperativity and a destabilization of the terminal G1-C20 (G1-C72 in tRNA)
base pair. Furthermore, the fact that this 3'-terminal imino proton is more
solvent-exposed at physiological temperature might be another indication
for the importance of the stability of the terminal base pair for specific
tRNA recognition.
ARTICLES
Structural studies on tRNA acceptor stem microhelices: exchange of the discriminator base A73 for G in human tRNALeu switches the acceptor specificity from leucine to serine possibly by decreasing the stability of the terminal G1-C72 base pair
Lehrstuhl fur Biopolymere, Universitat Bayreuth, D-95440 Bayreuth, Germany.
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