Probing the environment of lanthanide binding sites in yeast tRNA(Phe) by specific metal-ion-promoted cleavages.

Abstract

Specific yeast tRNA(Phe) hydrolysis brought about by europium ions has been studied in detail using the 32P-end-labeled tRNA and polyacrylamide gel electrophoresis. The dependence of the induced cleavages on pH, temperature and concentration of the europium ions has been determined. Europium hydrolyzes yeast tRNA(Phe) in the D-loop at phosphates 16 and 18, and the anticodon loop of phosphates 34 and 36. The two D-loop cuts are thought to take place from two distinct europium binding sites, while the two anticodon loop cleavages from a single site. Eight other members of the lanthanide series and ytrium give basically the same pattern of cleavages as europium. The specific cleavages taking place in the anticodon loop occur in an intramolecular mode from the lanthanide binding site that has not been found in yeast tRNA(Phe) crystal structure. It appears from the comparison of the europium-promoted cuts with those generated by magnesium and lead that the former two ions give more similar but not identical cleavage patterns. The usefulness of the specific cleavages induced by lanthanides for probing their own and magnesium binding sites in tRNA is discussed.