STRUCTURE AND AMINOACYLATION CAPACITIES OF TRANSFER-RNA TRANSCRIPTS CONTAINING DEOXYRIBONUCLEOTIDES

The contribution of the ribose 2'-hydroxyls to RNA structure and funct ion has been analyzed, but still remains controversial. In this work, we report the use of a mutant T7 RNA polymerase as a tool in RNA studi es, applied to the aspartate and methionine tRNA aminoacylation system s from yeast. Our approach consists of determining the effect of subst ituting natural ribonucleotides by deoxyribonucleotides in RNA and, th ereby, defining the subset of important 5'-hydroxyl groups. We show th at deoxyribose-containing RNA can be folded in a global conformation s imilar to that of natural RNA. Melting curves of tRNAs, obtained by te mperature-gradient gel electrophoresis, indicate that in deoxyribo-con taining molecules, the thermal stability of the tertiary network drops down, whereas the stability of the secondary structure remains unalte red. Nuclease footprinting reveals a significant increase in the acces sibility of both single- and double-stranded regions. As to the functi onality of the deoxyribose-containing tRNAs, their in vitro aminoacyla tion efficiency indicates striking differential effects depending upon the nature of the substituted ribonucleotides. Strongest decrease in charging occurs for yeast initiator tRNA(Met) transcripts containing d G or dC residues and for yeast tRNA(Asp) transcripts with dU or dG. In the aspartate system, the decreased aminoacylation capacities can be correlated with the substitution of the ribose moieties of U11 and G27 , disrupting two hydrogen bond contacts with the synthetase. Altogethe r, this suggests that specific 2'-hydroxyl groups in tRNAs can act as determinants specifying aminoacylation identity.