Conformation in solution of yeast tRNA(Asp) transcripts deprived of modified nucleotides

Abstract

A synthetic gene of yeast aspartic acid tRNA with a promoter for phage T7 RNA polymerase was cloned in Escherichia coli. The in vitro transcribed tRNA(Asp) molecules are deprived of modified nucleotides and retain their aspartylation capacity. The solution conformation of these molecules was mapped with chemical structural probes and compared to that of fully modified molecules. Significant differences in reactivities were observed in Pb2+ cleavage of the RNAs and in modification of the bases with dimethyl sulphate. The most striking result concerns C56, which becomes reactive in unmodified tRNA(Asp), indicating the disruption of the C56-G19 base pair involved in the D- and T-loop interaction. The chemical data indicate that unmodified tRNA(Asp) transcripts possess a relaxed conformation compared to that of the native tRNA. This conclusion is confirmed by thermal melting experiments. Thus it can be proposed that post-transcriptional modifications of nucleotides in tRNA stabilize the biologically active conformations in these molecules.