Enzymatic aminoacylation of sequence-specific RNA minihelices and hybrid duplexes with methionine

Abstract

RNA hairpin helices whose sequences are based on the acceptor stems of alanine and histidine tRNAs are specifically aminoacylated with their cognate amino acids. In these examples, major determinants for the identities of the respective tRNAs reside in the acceptor stem; the anticodon and other parts of the tRNA are dispensable for aminoacylation. In contrast, the anticodon is a major determinant for the identity of a methionine tRNA. RNA hairpin helices and hybrid duplexes that reconstruct the acceptor-T psi C stem and the acceptor stem, respectively, of methionine tRNA were investigated here for aminoacylation with methionine. Direct visualization of the aminoacylated RNA product on an acidic polyacrylamide gel by phosphor imaging demonstrated specific aminoacylation with substrates that contained as few as 7 base pairs. No aminoacylation with methionine was detected with several analogous RNA substrates whose sequences were based on noncognate tRNAs. While the efficiency of aminoacylation is reduced by orders of magnitude relative to methionine tRNA, the results establish that specific aminoacylation with methionine of small duplex substrates can be achieved without the anticodon or other domains of the tRNA. The results, combined with earlier studies, suggest a highly specific adaptation of the structures of aminoacyl-tRNA synthetases to the acceptor stems of their cognate tRNAs, resulting in a relationship between the nucleotide sequences/structures of small RNA duplexes and specific amino acids.