Kluyveromyces lactis γ-toxin, a ribonuclease that recognizes the anticodon stem loop of tRNA

摘要

Kluyveromyces lactis γ-toxin is a tRNA endonuclease that cleaves Saccharomyces cerevisiae , and between position 34 and position 35. All three substrate tRNAs carry a 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) residue at position 34 (wobble position) of which the mcm5 group is required for efficient cleavage. However, the different cleavage efficiencies of mcm5s2U₃₄-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 is cleaved at the same position as the natural substrate. In , the nucleotides U₃₄U₃₅C₃₆A₃₇C₃₈ 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 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.