2-thioribothymidine and ribothymidine

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.

Topics: Adenosine; Adenosine Triphosphate; Bacterial Proteins; Binding Sites; Carbon-Sulfur Lyases; Chromatography, High Pressure Liquid; Culture Media; Cysteine; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Lyases; Nucleic Acid Conformation; Nucleic Acid Denaturation; Oligonucleotides; Protein Binding; Recombination, Genetic; RNA; RNA Processing, Post-Transcriptional; RNA, Transfer; Sequence Analysis, RNA; Sulfates; Sulfurtransferases; Temperature; Thermus thermophilus; Thiouridine; tRNA Methyltransferases; Uridine

From Thermus thermophilus HB8 grown at 65 degrees C, two major tRNAIle species have been purified by column chromatography and polyacrylamide gel electrophoresis. The nucleotide sequence of one of these two tRNAIle1 species (tRNAIle1a) has been determined to be pGGGCGAUUAGCUCAGCUGmGUDAGAGCGCACGCCUGAUt6AAGCGUGAGm7GUCGGUGGs2T psi CAm1AGUCCACCAUCGCCCACCAOH. The nucleotide sequence of the other species (tRNAIle1b) is found to be the same as that of tRNAIle1a except for the modification in position 54; tRNAIle1a has s2T(54) while tRNAIle1b has T(54). The melting temperature of tRNAIle1a is as high as 86.2 degrees C while that of tRNAIle1b is 83.3 degrees C. The single replacement of an oxygen atom (2-carbonyl oxygen) of T(54) by a sulfur atom significantly contributes to the thermostability of the tRNAIle1a species. In addition, the methylation of G(18) and A(58) possibly contributes to the thermostability of T. thermophilus tRNAIle1a and tRNAIle1b species.

Topics: Base Sequence; Drug Stability; Kinetics; Nucleic Acid Conformation; Nucleic Acid Denaturation; RNA, Transfer, Amino Acyl; Thermodynamics; Thermus; Thiouridine; Uridine

An extreme thermophile, Thermus thermophilus HB 8, contains two types of tRNAs, T- and S2T-containing tRNAs. Their relative content changes depend on the growth temperature of the bacterial cells (1-3). To elucidate the reason why the extreme thermophile possesses the two types of tRNAs, an attempt was made to clarify how these tRNAs are utilized in in vivo protein synthetic systems of the bacteria cultured at different temperatures. First, a method was developed to isolate active polysomes from the thermophile cells cultured at 55 degrees C, 65 degrees C, and 77 degrees C. Then, tRNAs were separated from the polysomes and the T- and S2T-contents of the tRNAs were determined by HPLC. The relative content of S2T-tRNAs in the polysomes from 77 degrees C cells was much higher than that in bulk tRNAs from whole cells cultured at the same temperature, but the situation was reversed in 50 degrees C cells. These results clearly show that the protein synthetic systems of the thermophile have some selection mechanism to utilize either T- or S2T-containing tRNAs preferentially depending on the environmental temperature.

Topics: Bacterial Proteins; Centrifugation, Density Gradient; Chromatography, High Pressure Liquid; Polyribosomes; RNA, Bacterial; RNA, Transfer; Temperature; Thermus; Thiouridine; Uridine