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PubMed1988

Near-UV stress in Salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response.

We have examined the role of 4-thiouridine in the responses of Salmonella typhimurium to near-UV irradiation. Mutants lacking 4-thiouridine (nuv) and mutants defective in the synthesis of ppGpp (guanosine 5'-diphosphate-3'-diphosphate) (relA) were found to be sensitive to killing by near-UV. Near-UV induced the synthesis of a set of proteins that were not induced in the nuv mutant. Some of these proteins were identified as oxidative defense proteins, and others were identified as ppGpp-inducible proteins. Over 100-fold increases in ApppGpp (adenosine 5', 5"'-triphosphoguanosine-3"'-diphosphate, the adenylylated form of ppGpp) were observed in wild-type cells after near-UV irradiation but not in the 4-thiouridine-deficient mutant. These data support a model in which ppGpp and ApppGpp, a dinucleotide proposed to be synthesized by tRNA-aminoacyl synthetases as a response to the cross-linking of 4-thiouridine in tRNA by near-UV, induce the synthesis of proteins necessary for resistance to near-UV irradiation.

Bacterial Proteins↗
PubMed1987

Transfer RNA(5-methylaminomethyl-2-thiouridine)-methyltransferase from Escherichia coli K-12 has two enzymatic activities.

The tRNA(5-methylaminomethyl-2-thiouridine)-methyltransferase, which is involved in the biosynthesis of the modified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) present in the wobble position of some tRNAs, was purified close to homogeneity (95% purity). The molecular mass of the enzyme is 79,000 daltons. The enzyme activity has a pH optimum of 8.0-8.5, is inhibited by magnesium ions, and stimulated by ammonium ions. Two different intermediates in the biosynthesis of mnm5s2U34 are present in tRNA from the mutants trmC1 and trmC2. Unexpectedly, the product present in tRNA from trmC1 cells was identified by mass spectrometric and chromatographic analyses as 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), i.e. a more complex derivative than the final product mnm5s2U. The product present in tRNA from trmC2 cells was identified as 5-aminomethyl-2-thiouridine (nm5s2U). In the presence of S-adenosylmethionine the most purified enzyme fraction converts both cmnm5s2U34 and nm5s2U34 into mnm5s2U34. In the absence of S-adenosylmethionine, however, cmnm5s2U34 is converted into nm5s2U by this enzyme fraction. We conclude that the purified polypeptide has two enzymatic activities; one actually demodifies cmnm5s2U to nm5s2U and the other catalyzes the transfer of a methyl group from S-adenosylmethionine to nm5s2U, thus forming mnm5s2U. The sequential order of the biosynthesis of mnm5s2U34 is suggested to be: (Formula: see text). The molecular activity of the methyltransferase activity (nm5s2U34----mnm5s2U34) is 74 min-1, and the steady state concentration of the enzyme is only 78 molecules/genome equivalent in cells growing at a specific growth rate of 1.0/h.

Escherichia coli↗
PubMed2002

Transient reduction in the tRNA 4-thiouridine content induced by ultraviolet A during post-irradiation growth in Enterobacter cloacae.

The influence of previous exposure to ultraviolet-A radiation (UVA) was studied on the susceptibility of Enterobacter cloacae to undergo the growth delay effect. Comparison of growth curves corresponding to irradiated and control cells showed that a previous treatment with UVA almost abolished the growth delay effect. UV absorption spectra of tRNA, and reverse phase HPLC analysis of hydrolysed tRNA, demonstrated a low content of 4-thiouridine in E. cloacae cells grown after UVA exposure at low doses. Since 4-thiouridine is the UVA target responsible for initiation of growth delay, this observation explained the influence of previous exposure to UVA on the susceptibility of this organism to undergo growth delay. A similar but weaker alteration was found when Escherichia coli was assayed. The results suggest that, in addition to cross-linking with cytidine residues, the content of 4-thiouridine in tRNA may be modified by UVA by an unknown mechanism.

Dose-Response Relationship, Radiation↗
PubMed2003

A new route to 2'-O-alkyl-2-thiouridine derivatives via 4-O-protection of the uracil base and hybridization properties of oligonucleotides incorporating these modified nucleoside derivatives.

Oligonucleotides containing 2-thiouridine (s2U) in place of uridine form stable RNA duplexes with complementary RNAs. Particularly, this modified nucleoside has proved to recognize highly selectively adenosine, the genuine partner, without formation of a mismatched base pair with the guanosine counterpart. In this paper, we describe new methods for the synthesis of 2-thiouridine and various 2'-O-alkyl-2-thiouridine derivatives. Oligoribonucleotides having these modified nucleoside derivatives were synthesized, and their hybridization and structural properties were studied in detail by the 1H NMR analysis of these modified nucleosides and Tm experiments of RNA duplexes with their complementary RNA strands.

Magnetic Resonance Spectroscopy↗
PubMed2000

Synthesis of 4-thiouridine, 6-thioinosine, and 6-thioguanosine 3',5'-O-bisphosphates as donor molecules for RNA ligation and their application to the synthesis of photoactivatable TMG-capped U1 snRNA fragments.

4-Thiouridine, 6-thioguanosine, and 6-thioinosine 3',5'-bisphosphates (9, 20, and 28) were synthesized in good yields by considerably improved methods. In the former two compounds, uridine and 2-N-phenylacetylguanosine were converted via transient O-trimethylsilylation to the corresponding 4- and 6-O-benzenesulfonyl intermediates (2 and 13), which, in turn, were allowed to react with 2-cyanoethanethiol in the presence of N-methylpyrrolidine to give 4-thiouridine (3) and 2-N-phenylacetyl-6-thioguanosine derivatives (14), respectively. In situ dimethoxytritylation of these thionucleoside derivatives gave the 5'-masked products 4 and 15 in high overall yields from 1 and 11. 6-S-(2-Cyanoethyl)-5'-O-(4,4'-dimethoxytrityl)-6-thioinosine (23) was synthesized via substitution of the 5'-O-tritylated 6-chloropurine riboside derivative 22 with 2-cyanoethanethiol. These S-(2-cyanoethyl)thionucleosides were converted to the 2'-O-(tert-butyldimethylsilyl)ribonucleoside 3'-phosphoramidite derivatives 7, 18, and 26 or 3',5'-bisphosphate derivatives 8, 19, and 27. Treatment of 8, 19, and 27 with DBU gave thionucleoside 3',5'-bisphosphate derivatives 9, 20, and 28, which were found to be substrates of T4 RNA ligase. These thionucleoside 3',5'-bisphosphates were examined as donors for ligation with m3(2,2,7) G5'pppAmUmA, i.e., the 5'-terminal tetranucleotide fragment of U1 snRNA, The 4-thiouridine 3',5'-bisphosphate derivative 9 was found to serve as the most active substrate of T4 RNA ligase with a reaction efficiency of 96%.

Guanosine↗
PubMedDate not supplied

Efficient assessment of modified nucleoside stability under conditions of automated oligonucleotide synthesis: characterization of the oxidation and oxidative desulfurization of 2-thiouridine.

In order to efficiently assess the chemical stability of modified nucleosides to the reagents and conditions of automated oligonucleotide synthesis, we designed, developed and tested a scheme in which the modified nucleoside, directly attached to a solid support, is exposed to the cyclic chemistry of the instrument. Stability of 2-thiouridine against different oxidizers was investigated. Tertbutyl hydroperoxide (1 M) in anhydrous acetonitrile was a more effective oxidizer for the incorporation of 2-thiouridine into oligonucleotide chains than the same oxidizer in methylene chloride. Carbon tetrachloride/water in the presence of a basic catalyst was superior in maintaining the thiocarbonyl function, but its utility for RNA synthesis has yet to be fully tested, whereas 2-phenylsulfonyloxaziridine was a very efficient reagent for oxidative desulfurization of 2-thiouridine.

Acetonitriles↗
PubMed1998

Identification of a gene involved in the generation of 4-thiouridine in tRNA.

All organisms modify the bases of their RNA after transcription. Relatively little is known about the functions that these chemical alterations serve and, with very few exceptions, even less has been established regarding the enzymology involved. One modified base of known function is 4-thiouridine at position 8 of certain bacterial tRNAs, which serves as a photosensor for near-UV light. A gene involved in the conversion of uridine at position 8 into 4-thiouridine has been identified by genetic screening and its role in 4-thiouridine generation has been confirmed biochemically. This same gene, thiI , has recently been shown to play a role in thiamin biosynthesis. The purification and characteristics of the purified protein are also reported.

Animals↗
PubMed1999

Synthesis and properties of oligonucleotides containing 2'-O-methyl-2-thiouridine.

New methods to synthesize 2'-O-methyl-2-thiouridine and its phosphoramidite building block for incorporation into oligonucleotides were developed. Oligonucleotides containing 2'-O-methyl-2-thiouridine were expected to be favorable as antisense agents in several respects, i.e., nuclease resistance, stable RNA duplex formation, and exact base recognition. Therefore, to make them clear, we synthesized oligonucleotides having 2'-O-methyl-2-thiouridine and analyzed their properties in detail.

Indicators and Reagents↗
PubMed1999

Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis.

The gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique "P-loop" motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4-thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site-directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine.

Amide Synthases↗
PubMed1978

Incorporation of 6-thioguanosine and 4-thiouridine into RNA. Application to isolation of newly synthesised RNA by affinity chromatography.

Isolation of newly synthesised RNA can be achieved by treatment of cells in culture with 6-thioguanosine or 4-thiouridine followed by separation of thiol-containing RNA by affinity chromatography on mercurated cellulose columns. After short periods of treatment with 6-thioguanosine the proportion of RNA retained on mercurated cellulose is the same for both poly(A)-containing and poly(A)-free RNA, indicating similar incorporation of the drug into mRNA and rRNA. However, after longer periods of exposure, the cytotoxic effect of 6-thioguanosine results in diminished incorporation of radioactive uridine into RNA and of radioactive leucine into protein; this suggests that synthesis of both RNA and protein are impaired. On the other hand, even after long exposure to high concentrations of 4-thiouridine, the syntheses of RNA and protein are not significantly affected. Proteins synthesised after treatment of cells with 6-thioguanosine are less stable than proteins synthesised after treatment of cells with 4-thiouridine.

Animals↗
PubMed1993

A 5-4 pyrimidine-pyrimidone photoproduct produced from mixtures of thymine and 4-thiouridine irradiated with 334 nm light.

The nucleoside 4-thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near-UV light-induced growth delay and also mediates both photoprotection and near-UV cell killing in various bacterial strains. To investigate the photoreaction of 4-thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4-thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed-phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast-atom bombardment and electron-impact mass spectrometry, and 1H- and 13C-NMR spectroscopy, and have been identified as two diastereomers of 6-hydroxy-5-[1-(beta-D-erythro-pentofuranosyl)-4'-pyrimidin-2'- one]dihydrothymine (O6hThy[5-4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90 degrees C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.

Magnetic Resonance Spectroscopy↗
PubMed1982

Isolation of single-site Escherichia coli mutants deficient in thiamine and 4-thiouridine syntheses: identification of a nuvC mutant.

A method is described to rapidly select and classify many independent near-UV irradiation-resistant Escherichia coli mutants, which include tRNA modification and RNA synthesis control mutants. One class of these mutants was found to be simultaneously deficient in thiamine biosynthesis and in the ability to modify uridine in tRNA to 4-thiouridine, known to be the target for near-UV irradiation. These mutants were found to be unable to make thiazole, a thiamine precursor. The addition of thiazole restores the thiamine deficiency but does not render the cells near-UV irradiation sensitive. In vitro studies on one of these mutants indicated a deficiency in protein factor C (nuvC), required for the 4-thiouridine modification of tRNA. In P1 transduction, the thiazole marker cotransduced with the histidine marker, which places the thiazole marker between 42 and 46 min on the E. coli chromosome map. Both thiamine production and 4-thiouridine production were resumed by 87% of the spontaneous reversions, suggesting a single-point mutation. Our results indicate that we have isolated nuvC mutants and that the nuvC polypeptide is involved in two functions, tRNA modification and thiazole biosynthesis.

Chromosome Mapping↗
PubMedDate not supplied

[Using of 4-thiouridine and 4-thiothymidine for pyrimidine nucleoside phosphorylase studing].

4-Thiouridine and 4-thiothymidine were developed as efficient substrates for spectrophotometric determination of uridine phosphorylase and thymidine phosphorylase activity. 4-Thiouridine has maximum absorbance at 330 nm (pH 7.5). The change in extinction coefficient for 4-thiouridine/4-thiouracil, deltaepsilon is 3000 M(-1) x cm(-1). It appeared that 4-thiouridine is a good substrate for uridine phosphorylase with Michaelis-Menten constant 130 microM and kcat 49 s(-1). In the case of 4-thiothymidine/4-thiothymine deltaepsilon is even larger: 5000 M(-1) x cm(-1) at 336 nm.

Magnetic Resonance Spectroscopy↗
PubMed1977

[Mutants of Escherichia coli deficient in 4-thiouridine in which growth is insensitive to illumination at 365 nm].

Thirteen different mutants of E coli K12 selected for a reduced near ultra-violet induced growth delay have been isolated. The t-RNAs extracted from these clones have all a depressed content of 4-thiouridine. One of these mutants, called Nop has an almost negligible growth delay and completely lacks 4-thiouridine in its t-RNAs. Thus we provide genetic proofs that 4-thiouridine is the chromophore for growth delay.

Escherichia coli↗
PubMed1995

Aminoacylation of transfer RNAs with 2-thiouridine derivatives in the wobble position of the anticodon.

The first position or 'wobble base' in the anticodon of tRNAs is frequently the site of post-transcriptional modification. In Escherichia coli, glutamine, glutamate, and lysine tRNAs contain 2-thiouridine derivatives in this position, and the significance of these modifications has been under investigation since their discovery. Here we describe the investigations to link 2-thiouridine derivatives to aminoacylation of these tRNAs. The implications of these findings on the evolution of specificity of aminoacyl-tRNA synthetases and on translational regulation are also discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1994

Cross-linking of mRNA analogues containing 4-thiouridine residues on the 3'- or 5'-side of the coding triplet to the mRNA binding center of the human ribosome.

The interaction between mRNA and 18S rRNA within complexes of human placenta 80S ribosomes has been investigated by photochemical cross-linking experiments using mRNA analogues substituted with 4-thiouridine at specific locations. mRNA analogues 51 or 54 nucleotides long were prepared from synthetic DNA templates. These mRNA analogues contained either the sequence GGGACC (coding for glycine and threonine, respectively) or the single triplet GGG together with 2-4 4-thiouridine residues located at various positions with respect to the coding triplets. The products of cross-linking of the mRNA analogues to 18S rRNA within different model complexes without tRNA or in the presence of cognate tRNAs were analyzed by reverse transcription. Two cross-linking sites in the 18S rRNA were detected. The first site, U630, was cross-linked by mRNA 8' (s4U at +20, +22, +24, and +26), mRNA 9e' (s4U at -16, -18, and -20), and mRNA 10 (s4U at +4, +6, -1, and -3) but, unexpectedly, not with either mRNA 10b (s4U at +4 and +6) or mRNA 10c (s4U at -1 and -3). The second site, U1111/A1112, was cross-linked by mRNA 10 and mRNA 10c but not by any of the other mRNA analogues. There is significant tRNA dependence on cross-linking only for mRNA analogue 9e'. Both of the sites detected correspond to sites of mRNA cross-linking in Escherichia coli 16S rRNA.

Base Sequence↗
PubMed2003

MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli.

Thionucleosides are uniquely present in tRNA. In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34. The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance. Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli. We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E. coli. We also cloned mnmA from E. coli, and overproduced and purified the protein. Using a gel mobility shift assay, we show that MnmA binds to unmodified E. coli tRNA(Lys) with affinity in the low micromolar range. MnmA does not bind observably to the nonsubstrate E. coli tRNA(Phe). Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion. ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence. We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E. coli tRNA(Glu) as a substrate. The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS. HPLC analysis of thiolated tRNA digests using [(35)S]cysteine confirms that the product of the in vitro reaction is s(2)U. As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine. Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.

Aminopeptidases↗
PubMed1974

Photo-affinity labeling of tRNA binding sites in macromolecules. I. Linking of the phenacyl-p-azide of 4-thiouridine in (Escherichia coli) valyl-tRNA to 16S RNA at the ribosomal P site.

The phenacyl-p-azide of 4-thiouridine in (E. coli) tRNA(1) (Val) was prepared for use as a photo-affinity probe of tRNA binding sites on ribosomes. The derivatized tRNA was 90-100% as active as control tRNA for aminoacylation, nonenzymatic binding to the ribosomal P site, elongation factor Tu(EFTu)-dependent binding to the A site, EFTu-GTP-aa-tRNA ternary complex formation, and transfer of valine into polypeptide. Irradiation of p-azidophenacyl-[(3)H]valyl-tRNA bound noncovalently to the ribosomal P site resulted in covalent attachment of 15-20% of the noncovalently bound tRNA to the ribosomes. The linking occurred exclusively to the 16S RNA of the 30S ribosomal subunit, thus suggesting that the region of the ribosome within 9 A of the 4-thiouridine of tRNA, when it is bound in the P site, is solely 16S RNA.

Binding Sites↗