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

Author： Lu Jian Esberg Anders Huang Bo Byström Anders S.

ISSN： 1362-4962

Source： Nucleic Acids Research, Vol.36, Iss.4, 2008-03, pp. : 1072-1080

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Abstract

Kluyveromyces lactis -toxin is a tRNA endonuclease that cleaves Saccharomyces cerevisiae ${tRNA}_{{mcm}^{5} s^{2} UUC}^{Glu 3}$ , ${tRNA}_{{mcm}^{5} s^{2} UUU}^{Lys}$ and ${tRNA}_{{mcm}^{5} s^{2} UUG}^{Gln}$ 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 -toxin. A synthetic minihelix RNA corresponding to the anticodon stem loop (ASL) of the natural substrate ${tRNA}_{{mcm}^{5} s^{2} UUC}^{Glu 3}$ is cleaved at the same position as the natural substrate. In ${ASL}_{UUC}^{Glu 3}$ , the nucleotides U₃₄U₃₅C₃₆A₃₇C₃₈ are required for optimal -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 ${tRNA}_{UUC}^{Glu}$ 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.