Kluyveromyces lactis γ-toxin is a tRNA endonuclease that cleaves Saccharomyces cerevisiae tRNAmcm5s2UUCGlu3, tRNAmcm5s2UUULys and tRNAmcm5s2UUG Gln between position 34 and position 35. All three substrate tRNAs carry a 5-methoxycarbonylmethyl-2-thiouridine (mcm5 s2U) residue at position 34 (wobble position) of which the mcm5 group is required for efficient cleavage. However, the different cleavage efficiencies of mcm5s2 U34-containing tRNAs suggest that additional features of these tRNAs affect cleavage. In the present study, we show that a stable anticodon stem and the anticodon loop are the minimal requirements for cleavage by γ-toxin. A synthetic minihelix RNA corresponding to the anticodon stem loop (ASL) of the natural substrate tRNAmcm5s2UUCGlu3 is cleaved at the same position as the natural substrate. In ASLUUCGlu3, the nucleotides U34U35C36A37 C38 are required for optimal γ-toxin cleavage, whereas a purine at position 32 or a G in position 33 dramatically reduces the cleavage of the ASL. Comparing modified and partially modified forms of E. coli and yeast tRNAUUCGlu reinforced the strong stimulatory effects of the mcm5 group, revealed a weak positive effect of the s2 group and a negative effect of the bacterial 5-methylaminomethyl (mnm5) group. The data underscore the high specificity of this yeast tRNA toxin.
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