CLEAVAGES INDUCED BY DIFFERENT METAL-IONS IN YEAST TRNA(PHE) U59C60 MUTANTS

The U59 and C60 residues, which form the strong Pb(II) ion binding sit e in yeast tRNA(Phe), were systematically mutated, and the effects of individual mutations on cleavages induced by various metal ions were a nalyzed. It turned out that the presence of C60 is essential for effic ient cleavage of the D-loop by Pb(II), Eu(III), and Mg(II) ions. On th e other hand, manganese ions were capable of cleaving these mutants wi th an efficiency similar to that observed for the wild type transcript . Moreover, in all C60 mutants, the main Mn(II) cleavage site was shif ted by one phosphate from P16 to P17. Mutations of U59 did not affect so dramatically the efficiency and specificity of the D-loop hydrolysi s induced by all studied metal ions. In the G59C60 mutant cleaved by P b(II) ions, new cuts took place in the T-stem at P63-P65. Also, the C6 0 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) cleava ge at P61 was suppressed. Nevertheless, the changes in overall tRNA st ructure resulting from U59 and C60 mutations were rather subtle. The s tudied mutants showed S-1 and V-1 nuclease digestion patterns practica lly indistinguishable from those observed in the wild type transcript. The metal ions are shown to be well-suited for monitoring the local c hanges in the structure of the investigated tRNA variants and when use d as a set of probes can give a more complete picture of changes that occur in transcripts as a result of a mutation.