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J. Biol. Chem., Vol. 281, Issue 4, 2104-2113, January 27, 2006

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Temperature-dependent Biosynthesis of 2-Thioribothymidine of Thermus thermophilus tRNA^*

Naoki Shigi, Tsutomu Suzuki, Takaho Terada¶||, Mikako Shirouzu¶||, Shigeyuki Yokoyama¶||**, and Kimitsuna Watanabe1

From the Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, 2-42 Aomi, Koto-ku, Tokyo 135-0064, Japan, the Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, ^¶RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Yokohama 230-0045, Japan, ^||RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, and the ^**Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

2-Thioribothymidine (s²T) is a modified nucleoside of U, specifically found at position 54 of tRNAs from extreme thermophilic microorganisms. The function of the 2-thiocarbonyl group of s²T54 is thermostabilization of the three-dimensional structure of tRNA; however, its biosynthesis has not been clarified until now. Using an in vivo tRNA labeling experiment, we demonstrate that the sulfur atom of s²T in tRNA is derived from cysteine or sulfate. We attempted to reconstitute 2-thiolation of s²T in vitro, using a cell extract of Thermus thermophilus. Specific 2-thiolation of ribothymidine, at position 54, was observed in vitro, in the presence of ATP. Using this assay, we found a strong temperature dependence of the 2-thiolation reaction in vitro as well as expression of 2-thiolation enzymes in vivo. These results suggest that the variable content of s²T in vivo at different temperatures may be explained by the above characteristics of the enzymes responsible for the 2-thiolation reaction. Furthermore, we found that another posttranscriptionally modified nucleoside, 1-methyladenosine at position 58, is required for the efficient 2-thiolation of ribothymidine 54 both in vivo and in vitro.

Received for publication, October 3, 2005 , and in revised form, November 28, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-3-3599-8106; Fax: 81-3-5530-2064; E-mail: kwatanab{at}jbirc.aist.go.jp.

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