ACTIVATION OF MICROHELIX CHARGING BY LOCALIZED HELIX DESTABILIZATION

We report that aminoacylation of minimal RNA helical substrates is enh anced by mismatched or unpaired nucleotides at the first position in t he helix. Previously, we demonstrated that the class I methionyl-tRNA synthetase aminoacylates RNA microhelices based on the acceptor stem o f initiator and elongator tRNAs with greatly reduced efficiency relati ve to full-length tRNA substrates. The cocrystal structure of the clas s I glutaminyl-tRNA synthetase with tRNA(Gln) revealed an uncoupling o f the first (1.72) base pair of tRNA(Gln), and tRNA(Met) was proposed by others to have a similar base-pair uncoupling when bound to methion yl-tRNA synthetase. Because the anticodon is important for efficient c harging of methionine tRNA, we thought that 1.72 distortion is probabl y effected by the synthetase-anticodon interaction. Small RNA substrat es (minihelices, microhelices, and duplexes) are devoid of the anticod on triplet and may, therefore, be inefficiently aminoacylated because of a lack of anticodon-triggered acceptor stem distortion. To test thi s hypothesis, we constructed microhelices that vary in their ability t o form a 1.72 base pair. The results of kinetic assays show that micro helix aminoacylation is activated by destabilization of this terminal base pair. The largest effect is seen when one of the two nucleotides of the pair is completely deleted. Activation of aminoacylation is als o seen with the analogous deletion in a minihelix substrate for the cl osely related isoleucine enzyme. Thus, for at least the methionine and isoleucine systems, a built-in helix destabilization compensates in p art for the lack of presumptive anticodon-induced acceptor stem distor tion.