Tertiary structure of tRNAs in solution monitored by phosphodiester modification with ethylnitrosourea

Abstract

The alkylation by ethylnitrosourea of phosphodiester bonds in yeast tRNAPhe, tRNAVal and in Escherichia coli tRNAGlu, tRNAfMet, tRNAmMet and tRNAPhe was investigated under various conditions. In unfolded tRNAs the reactivities of phosphates in various positions toward the reagent were similar. In the folded tRNAs remarkable differences in reactivities of phosphates located in various positions of the molecules were observed. In yeast and E. coli tRNAPhe, reactivities of phosphates in positions 9, 10, 11, 19, 49, 58, 59 and 60 were found to be strongly decreased. Some decrease in reactivity was observed for phosphates 23 and 24. Spermine and ethidium bromide did not influence the pattern of phosphate alkylation in the T psi C arm of yeast tRNAPhe. Our solution results fit with the crystal structure of tRNAPhe with respect to the potential availability of the phosphates in this tRNA to solvent as shown by others. Judging from the pattern of phosphate reactivities, the structure of E. coli tRNAPhe is very similar to that of yeast tRNAPhe. Upon thermal denaturation of the yeast tRNAPhe, the reactivity of the low-reactive phosphates increased, demonstrating a cooperative melting curve. A comparison of the patterns of phosphate alkylation in several tRNAs, essentially in their T psi C arms, revealed a striking similarity, suggesting that the folding of these tRNAs is essentially similar.