Temperature-dependent Biosynthesis of 2-Thioribothymidine ofThermus thermophilustRNA
Journal of Biological Chemistry
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
... 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. NH 4 Cl, ZnSO 4 -7H 2 O to ZnCl 2 , CuSO 4 -5H 2 O to CuCl 2 , and FeSO 4 -7H 2 O to FeCl 3 -6H 2 O. VOSO 4 -xH 2 O, biotin, and thiamine-HCl were not used as supplements in MMϪS medium. In Vivo Labeling of RNA-Cells were cultivated overnight in 10 ml of MM at 70°C, centrifuged, and washed with MMϪS medium. They were then incubated with shaking at 70°C for 1 h in 10 ml of MM-S supplemented with 0.27 mCi of [ 35 S]methionine, 0.41 mM cysteine, and 0.4 mM sodium sulfate for the methionine labeling; 0.27 mCi of [ 35 S]cysteine, 0.33 mM methionine, and 0.4 mM sodium sulfate for the cysteine labeling; and 0.27 mCi of [ 35 S]sodium sulfate, 0.33 mM methionine, and 0.41 mM cysteine for the sulfate labeling. 35 S-Labeled compounds were purchased from American Radiolabeled Chemicals. Total RNA was extracted from the cells using ISOGEN (Wako). For alkaline treatment of tRNA, total RNA was incubated at 37°C for 1 h in 100 mM HEPES-KOH (pH 9.0). Then total RNA was separated by 10% PAGE containing 7 M urea, and the gel was dried and exposed to an imaging plate, followed by analysis using a BAS5000 bioimaging analyzer (Fuji Photo Systems). . 2 The abbreviations used are: s 2 T, 2-thioribothymidine; rT, ribothymidine; T m , melting temperature; s 4 U, 4-thiouridine; LC/MS, liquid chromatography/mass spectroscopy; , pseudouridine; m 1 A, 1-methyladenosine; APM, [(N-acryloylamino)phenyl]mercuric chloride; mnm 5 s 2 U, 5-methylaminomethyl-2-thiouridine; ms 2 i 6 A, 6-isopentenyl-2-methylthioadenosine; s 2 C, 2-thiocytidine; ms 2 io 6 A, 6-(4-hydroxyisopentenyl)-2methylthioadenosine; Gm, 2Ј-O-methylguanosine; HPLC, high performance liquid chromatography.