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

Abstract

2-Thioribothymidine (s 2 T) 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 s 2 T54 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 s 2 T in tRNA is derived from cysteine or sulfate. We attempted to reconstitute 2-thiolation of s 2 T 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 s 2 T 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.Post-transcriptional modification is a characteristic feature of RNA molecules. In transfer RNA, post-transcriptional modification plays various roles that are required for the translation process, including fidelity control of codon recognition, reading frame maintenance, and stabilization of the tertiary tRNA structure (1).2-thioribothymidine (s 2 T) 2 is a 2-thiolated derivative of 5-methyluridine (ribothymidine (rT)), located at position 54 of Thermus thermophilus tRNA (2), and has been shown to stabilize tRNA structure in a high temperature environment (3). In the extreme thermophilic eubacteria, Thermus thermophilus (4), and the archaea, Pyrococcus furiosus (5), the 2-thiolation level of rT54 in tRNA increases with cultivation temperature; the melting temperature (T m ) of the tRNA increases concomitantly with incremental increases in s 2 T content. These findings indicate that 2-thiolation of rT54 is responsible for the thermostability of thermophile tRNA at a variety of cultivation temperatures, thereby ensuring the adaptation of the protein synthesis machinery to specific thermal environments.However, the body of knowledge regarding s 2 T biosynthesis is limited. First, the enzymes responsible for the tRNA modification and the genes involved in the 2-thiolation reaction have not been identified to date. Previously, we demonstrated that the tRNA structural elements required for 2-thiolation are the conserved bases in the TC loop and the structure created by those bases (6). In this report, we investigated the sulfur donor for s 2 T biosynthesis in vivo. We also examined the 2-thiolation reaction in vitro, in order to characterize the temperature dependence of this reaction, and considered the contribution of neighboring nucleoside modifications to the efficiency of 2-thiolation of rT54, both in vivo and in vitro. ...

The tRNA Recognition Mechanism of Folate/FAD-dependent tRNA Methyltransferase (TrmFO)

“…This observed effect is probably caused by the reinforcement of the U54-A58 tertiary base pair. The m 1 A58 modification is a positive determinant for the 2-thiouridylation complex (TtuA, TtuB, TtuC, and the other protein factors), which catalyzes s 2 U54 formation (8,9). Therefore, our current results and other reports (8,9) (47), we found that the Arg-97, Lys-282, and Lys-287 residues in TrmFO are important for the methyl transfer reaction.…”

“…The m 1 A58 modification is a positive determinant for the 2-thiouridylation complex (TtuA, TtuB, TtuC, and the other protein factors), which catalyzes s 2 U54 formation (8,9). Therefore, our current results and other reports (8,9) (47), we found that the Arg-97, Lys-282, and Lys-287 residues in TrmFO are important for the methyl transfer reaction. Based on the current and previous experimental results, we constructed the docking model of TrmFO and T-arm of yeast tRNA Phe (Fig.…”

Trmt61B is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial tRNAs

“…In Thermus thermophilus, inactivation of TrmI induces a thermosensitive phenotype (Droogmans et al 2003). Since TrmI disruption strongly reduced 2-thiolation of 5-methyl-2-thiouridine (s 2 T) at position 54 (Shigi et al 2006b), the decreased thermo-tolerance observed in the DTrmI strain can be partly explained by the lack of 2-thiolation of s 2 T54, a key modification required for tRNA thermostability (Yokoyama et al 1987;Shigi et al 2006a).…”

Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA

“…, and 2-methylthio-N6-alkyladenosine (2). Most of these modifications are found in transfer RNA (tRNA), where they have various important cellular roles, such as controlling the fidelity of translation (3)(4)(5), recognizing aminoacyltRNA synthetase (6), sensing UV radiation stress (7), and stabilizing the ternary structure of tRNA for growth at high temperatures (8)(9)(10).…”