thiouridine and 2-thioribothymidine

4-Thiouridine (s

Topics: Biotin; RNA; RNA, Transfer; Saccharomyces cerevisiae; Thiouridine

TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s

Topics: Adenosine Triphosphate; Bacterial Proteins; Crystallography, X-Ray; Electron Spin Resonance Spectroscopy; Iron-Sulfur Proteins; Models, Molecular; Multigene Family; Mutation; Protein Binding; Protein Conformation; RNA, Transfer; Structure-Activity Relationship; Substrate Specificity; Sulfurtransferases; Thermus thermophilus; Thiouridine

Ribozyme-catalyzed RNA polymerization is inefficient and error prone. Here we demonstrate that two alternative bases, 2-thio-uridine (s(2)U) and 2-thio-ribo-thymidine (s(2)T), improve the rate and fidelity of ribozyme catalyzed nucleotide addition as NTP substrates and as template bases. We also demonstrate the functionality of s(2)U and s(2)T-containing ribozymes.

Topics: Biocatalysis; Kinetics; Reverse Transcription; RNA; RNA, Catalytic; Thiouridine; Transcription, Genetic

The ubiquitin-like protein TtuB is a sulfur carrier for the biosynthesis of 2-thioribothymidine (s

Topics: Amino Acid Sequence; Amino Acid Substitution; Bacterial Proteins; Cations, Divalent; Cloning, Molecular; Crystallization; Crystallography, X-Ray; Cysteine; Escherichia coli; Gene Expression; Genetic Vectors; Glycine; Mutation; Recombinant Proteins; RNA, Transfer; Thermus thermophilus; Thiouridine; X-Ray Diffraction; Zinc

Incorporation of a sulfur atom into 2-thioribothymidine (s

Topics: Amino Acid Sequence; Bacterial Proteins; Escherichia coli; Gene Expression; Molecular Mimicry; Recombinant Proteins; RNA, Bacterial; RNA, Transfer; Sequence Alignment; Sequence Homology, Amino Acid; Sulfurtransferases; Thermus thermophilus; Thiosulfate Sulfurtransferase; Thiouridine

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

The interactions of tRNA species with aminoacyl-tRNA synthetases and polypeptide chain elongation factor Tu from Thermus thermophilus HB8 were studied by the analyses mainly of the circular dichroism band of 2-thioribothymidine in position 54 of T. thermophilus tRNA species.

Topics: Amino Acyl-tRNA Synthetases; Circular Dichroism; Glutamates; Glutamic Acid; Nucleic Acid Conformation; Peptide Elongation Factor Tu; Protein Conformation; RNA, Transfer; Thermus; Thiouridine

35S incorporation studies showed that Candida tropicalis tRNA contained two thionucleosides, one of which was identified as 5-methyl-2-thiouridine. The other thionucleoside was alkali labile, and it appeared to be an ester. Pulse-chase experiments suggested that the two thionucleosides were structurally related. 5-Methyl-2-thiouridine was present in one of the lysine tRNAs. This is the first report of the presence of this nucleoside in a yeast tRNA.

Topics: Candida; RNA, Fungal; RNA, Transfer; RNA, Transfer, Amino Acyl; Thionucleosides; Thiouridine

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

1H-NMR analyses have been made on the conformations of 2-thioribothymidine (s2T), 2-thiodeoxyribothymidine (s2dT), as well as ribothymidine (T) and deoxyribothymidine (dT). s2T and s2dT exclusively take the anti form rather than the syn form. The C3'-endo-gg form of the sugar moiety is remarkably stabilized on modification of T to s2T, but not on modification of dT to s2dT. The steric effects of the 2-thiocarbonyl group and the 2'-hydroxyl group cause the rigidity of the C3'-endo-gg form of s2T. Such rigidity of s2T probably contributes to the thermostability of 2-thiopyrimidine polyribonucleotides and extreme thermophile tRNAs.

Topics: Eubacterium; Hot Temperature; Magnetic Resonance Spectroscopy; Nucleic Acid Conformation; RNA, Bacterial; RNA, Transfer; Structure-Activity Relationship; Thiouridine

For the extreme thermophile Thermus thermophilus HB 8, a positive correlation was observed among the growth temperatures of the cells, the melting temperature, and the 2-thioribothymidine (s2T) content of tRNA extracted from cells grown at various temperatures [Watanabe, K., Shinma, M., Oshima, T., & Nishimura, S. (1976) Biochem. Biophys. Res. Commun. 72, 1137-1144]. On the basis of these observations, studies were carried out from which the following results were obtained. (1) Both RNase T1 and U2 digestions of tRNA gave only four fragments containing s2T or T: s2T psi CGp, s2T psi CAp, T psi CGp, and T psi CAp. For the different growth temperatures, the ratio of the content of s2T psi CGp plus s2T psi CAp to that of T psi CGp plus T psi CAp was almost the same as that of the s2Tp to Tp content reported previously. (2) The midpoint of the s2T-specific circular dichroism spectral change induced by heat was constant for all tRNAs extracted from cells grown at various temperatures, suggesting that the s2T-containing tRNAs melt at about the same temperature, which is independent of the growth temperature of cells. (3) s2T-containing tRNA was separated from the T-containing counterpart quantitatively by a specific modification of s2T with bromoaceto-2,4-dinitroanilide followed by BD-cellulose column chromatography. The molar ratio of the s2T- and T-containing tRNAs was also similar to that of s2Tp to Tp as mentioned above. These results demonstrate that T. thermophilus cells have both s2T- and T-containing tRNAs, whose relative content is controlled by the growth temperature. This phenomenon may be necessary to enable the thermophile to adapt to higher temperatures.

Topics: RNA, Transfer; Temperature; Thermus; Thiouridine

Topics: Chemical Phenomena; Chemistry; Circular Dichroism; Escherichia coli; Hydrogen Peroxide; Kinetics; Nucleic Acid Conformation; RNA, Transfer; Thermus; Thiouridine

Topics: Circular Dichroism; Drug Stability; Hot Temperature; Magnetic Resonance Spectroscopy; Nucleic Acid Conformation; RNA, Transfer; Thermus; Thiouridine