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

Abstract

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.

Identification of the magnesium, europium and lead binding sites in E. coli and lupine tRNAPhe by specific metal ion‐induced cleavages

“…The cleavages occurring in tRNA Phe from yeast, tRNA TM hydrolysis obtained in this experiment is in perfect agreement with that observed earlier in the range of low concentrations of Pb ions [14]. The lupine tRNA vh" having the same nucleotide sequence as yeast tRNA eh" in both the T-loop and Dloop, i.e.…”

“…The absence of any Pb-induced cleavages in that region in yeast tRNA Phe [14][15][16] was confirmed in the present study. There are also no europium and magnesium-induced cleavages in the variable loop.…”

“…The reaction temperature, pH of the buffer, concentrations of metal ions and reaction time are specified in the figure legends. Prior to reactions, tRNAs were subjected to the standard denaturation-renaturation procedure [14].…”

“…Here, we describe the cleavage patterns and analyze the Pb, Eu and Mg binding sites in E. coli and lupine tRNA Phe. The results are compared directly with those obtained for yeast tRNA phe [12][13][14][15][16]. Abbreviations: Y-base, et-(carboxyamino)-4,9-dihydro-4,6-dimethyl-9-oxo-lH-imidazolo[1,2-a]purine-7-butyric acid dimethyl ester; o2yW, a-(carboxyamino)-4,9-dihydro-#-hydroperoxy-4,6-dimethyl-9-oxo-1H-imidazoio[ 1,2-a]purine-7-butyric acid dimethyl ester; ms2i6A, 2-methylthio-A#-(A2-isopentenyl)adenosine…”

Imperfect CAG Repeats Form Diverse Structures in SCA1 Transcripts

“…Among the single-strand specific nucleases used were the nucleases S1 and Mung Bean showing little sequence specificity, RNase T2 cutting all internucleotide bonds but showing some preference for those after A-residues and G-specific RNase T1 (31,32). The metal ion probes were represented by lead ions that cleave singlestranded RNA sections and relaxed parts of duplex structures (33)(34)(35). Also, the short oligodeoxynucleotide 5Ј-TGCTGCT (ctg-ODN), as well as the ODN libraries (6-mer and 7-mer) with a randomized sequence were used together with Escherichia coli RNase H, which executed RNA cleavages at the sites of RNA/DNA hybrids.…”