Characterization of the lead(II)-induced cleavages in tRNAs in solution and effect of the Y-base removal in yeast tRNAPhe.

The specificity of lead(II)-induced hydrolysis of yeast tRNA(Phe) was studied as a function of concentration of Pb2+ ions. The major cut was localized in the D-loop and minor cleavages were detected in the anticodon and T-loops at high metal ion concentration. The effects of pH, temperature, and urea were also analyzed, revealing a basically unchanged specificity of hydrolysis. In the isolated 5'-half-molecule of yeast tRNAPhe not cut was found in the D-loop, indicating its stringent dependence on T-D-loop interaction. Comparison of hydrolysis patterns and efficiencies observed in yeast tRNA(Phe) with those found in other tRNAs suggests that the presence of a U59-C60 sequence in the T-loop is responsible for the highly efficient and specific hydrolysis in the spatially close region of the D-loop. The efficiencies of D-loop cleavage in intact yeast tRNA(Phe) and in tRNA(Phe) deprived of the Y base next to the anticodon were also compared at various Pb2+ ion concentrations. Kinetics of the D-loop hydrolysis analyzed at 0, 25, and 37 degrees C showed a 6 times higher susceptibility of tRNA(Phe) minus Y base (tRNA(Phe)-Y) to lead(II)-induced hydrolysis than in tRNA(Phe). The observed effect is discussed in terms of a long-distance conformational transition in the region of the interacting D- and T-loops triggered by the Y-base excision.