EVIDENCE FOR A CONSERVED RELATIONSHIP BETWEEN AN ACCEPTOR STEM AND A TRANSFER-RNA FOR AMINOACYLATION

The anticodon-independent aminoacylation of RNA hairpin helices that r econstruct tRNA acceptor stems has been demonstrated for at least 10 a minoacyl-tRNA synthetases. For Escherichia coli cysteine tRNA syntheta se, the specificity of aminoacylation of the acceptor stem is determin ed by the U73 nucleotide adjacent to the amino acid attachment site. B ecause U73 is present in all known cysteine tRNAs, we investigated the ability of the E. coli cysteine enzyme to aminoacylate a heterologous acceptor stem. We show here that a minihelix(Cys) based on the accept or-T Psi C stem of yeast tRNA(Cys) is a substrate for the E. coli enzy me, and that aminoacylation of this minihelix is dependent on U73. Add itionally, we identify two base pairs in the acceptor stem that quanti tatively convert the E. coli acceptor stem to the yeast acceptor stem. The influence of U73 and these two base pairs is completely retained in the full-length tRNA. This suggests a conserved relationship betwee n the acceptor stem alone and the acceptor stem in the context of a tR NA for aminoacylation with cysteine. However, the primary determinant in the species-specific aminoacylation of the E. coli and yeast cystei ne tRNAs is a tertiary base pair at position 15:48 outside of the acce ptor stem. Although E. coli tRNA(Cys) has unusual G15:G48 tertiary bas e pair, yeast tRNA(Cys) has more common G15:C48 that prevents efficien t aminoacylation of yeast tRNA(Cys) by the E. coli enzyme. Our results support the notion that the conserved determinants in the acceptor st em may have provided a primordial code for cysteine, and that this cod e evolved in the development of tRNA sequences to include an additiona l element, such as the 15:48 tertiary base pair.