Cleavages induced by different metal ions in yeast tRNA(Phe) U59C60 mutants.

Abstract

The U59 and C60 residues, which form the strong Pb(II) ion binding site in yeast tRNA(Phe), were systematically mutated, and the effects of individual mutations on cleavages induced by various metal ions were analyzed. It turned out that the presence of C60 is essential for efficient cleavage of the D-loop by Pb(II), Eu(III), and Mg(II)ions. On the other hand, manganese ions were capable of cleaving these mutants with an efficiency similar to that observed for the wild type transcript. Moreover, in all C60 mutants, the main Mn(II) cleavage site was shifted by one phosphate from P16 to P17. Mutations of U59 did not affect so dramatically the efficiency and specificity of the D-loop hydrolysis induced by all studied metal ions. In the G59C60 mutant cleaved by Pb(II) ions, new cuts took place in the T-stem at P63-P65. Also, the C60 mutants were cleaved more strongly at P49 by Pb(II) ions. In G59C60 and A59C60 as well as in all C60 mutants, the Mg(II) and Mn(II) cleavage at P61 was suppressed. Nevertheless, the changes in overall tRNA structure resulting from U59 and C60 mutations were rather subtle. The studied mutants showed S1 and V1 nuclease digestion patterns practically indistinguishable from those observed in the wild type transcript. The metal ions are shown to be well-suited for monitoring the local changes in the structure of the investigated tRNA variants and when used as a set of probes can give a more complete picture of changes that occur in transcripts as a result of a mutation.