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Nucleic Acids Research Advance Access originally published online on December 20, 2007
Nucleic Acids Research 2008 36(4):1072-1080; doi:10.1093/nar/gkm1121
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Nucleic Acids Research, 2008, Vol. 36, No. 4 1072-1080
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Kluyveromyces lactis {gamma}-toxin, a ribonuclease that recognizes the anticodon stem loop of tRNA

Jian Lu, Anders Esberg, Bo Huang and Anders S. Byström*

Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden

*To whom correspondence should be addressed. Tel: +46 90 7856764; Fax: +46 90 772630; Email: anders.bystrom{at}molbiol.umu.se

Received October 6, 2007. Revised November 30, 2007. Accepted December 1, 2007.

Kluyveromyces lactis {gamma}-toxin is a tRNA endonuclease that cleaves Saccharomyces cerevisiae Formula, Formula and Formula 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 {gamma}-toxin. A synthetic minihelix RNA corresponding to the anticodon stem loop (ASL) of the natural substrate Formula is cleaved at the same position as the natural substrate. In Formula, the nucleotides U34U35C36A37C38 are required for optimal {gamma}-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 Formula 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.

Present address: Jian Lu, Laboratory of Molecular Gerontology, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA


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