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.