Publications

• Ribonucleic acid sulfurtransferase from Bacillus subtilis W168. Sulfuration with beta-mercaptopyruvate and properties of the enzyme system.

  Wong T.W., Weiss S.B., Eliceiri G.L., Bryant J.

  Biochemistry 9:2376-2386(1970) [PubMed] [EuropePMC]

• The biosynthesis of 4-thiouridylate. Separation and purification of two enzymes in the transfer ribonucleic acid-sulfurtransferase system.

  Abrell J.W., Kaufman E.E., Lipsett M.N.

  J Biol Chem 246:294-301(1971) [PubMed] [EuropePMC]

• The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA.

  Mueller E.G., Palenchar P.M., Buck C.J.

  The enzyme ThiI is common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli ThiI) found in this motif is essential for activ ... >> More

  The enzyme ThiI is common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli ThiI) found in this motif is essential for activity (Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J., and Mueller, E. G. (2000) J. Biol. Chem. 275, 8283-8286). In light of that finding and the report of the involvement of the protein IscS in the reaction (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568), we proposed two mechanisms for the sulfur transfer mediated by ThiI, and both suggested possible involvement of the thiol group of another cysteine residue in ThiI. We have now substituted each of the cysteine residues with alanine and characterized the effect on activity in vivo and in vitro. Cys-108 and Cys-202 were converted to alanine with no significant effect on ThiI activity, and C207A ThiI was only mildly impaired. Substitution of Cys-344, the only cysteine residue conserved among all sequenced ThiI, resulted in the loss of function in vivo and a 2700-fold reduction in activity measured in vitro. We also examined the possibility that ThiI contains an iron-sulfur cluster or disulfide bonds in the resting state, and we found no evidence to support the presence of either species. We propose that Cys-344 forms a disulfide bond with Cys-456 during turnover, and we present evidence that a disulfide bond can form between these two residues in native ThiI and that disulfide bonds do form in ThiI during turnover. We also discuss the relevance of these findings to the biosynthesis of thiamin and iron-sulfur clusters. << Less

  J. Biol. Chem. 276:33588-33595(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Crystal structure of a 4-thiouridine synthetase-RNA complex reveals specificity of tRNA U8 modification.

  Neumann P., Lakomek K., Naumann P.T., Erwin W.M., Lauhon C.T., Ficner R.

  In prokaryotes and archaea transfer ribonucleic acid (tRNA) stability as well as cellular UV protection relies on the post-transcriptional modification of uracil at position 8 (U8) of tRNAs by the 4-thiouridine synthetase ThiI. Here, we report three crystal structures of ThiI from Thermotoga marit ... >> More

  In prokaryotes and archaea transfer ribonucleic acid (tRNA) stability as well as cellular UV protection relies on the post-transcriptional modification of uracil at position 8 (U8) of tRNAs by the 4-thiouridine synthetase ThiI. Here, we report three crystal structures of ThiI from Thermotoga maritima in complex with a truncated tRNA. The RNA is mainly bound by the N-terminal ferredoxin-like domain (NFLD) and the THUMP domain of one subunit within the ThiI homo-dimer thereby positioning the U8 close to the catalytic center in the pyrophosphatase domain of the other subunit. The recognition of the 3'-CCA end by the THUMP domain yields a molecular ruler defining the specificity for U8 thiolation. This first structure of a THUMP/NFLD-RNA complex might serve as paradigm for the RNA recognition by THUMP domains of other proteins. The ternary ThiI-RNA-ATP complex shows no significant structural changes due to adenosine triphosphate (ATP) binding, but two different states of active site loops are observed independent of the nucleotide loading state. Thereby conformational changes of the active site are coupled with conformational changes of the bound RNA. The ThiI-RNA complex structures indicate that full-length tRNA has to adopt a non-canonical conformation upon binding to ThiI. << Less

  Nucleic Acids Res 42:6673-6685(2014) [PubMed] [EuropePMC]

• RNA thiolase: the enzymatic transfer of sulfur from cysteine to sRNA in Escherichia coli extracts.

  Hayward R.S., Weiss S.B.

  Proc Natl Acad Sci U S A 55:1161-1168(1966) [PubMed] [EuropePMC]

• Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA.

  Kambampati R., Lauhon C.T.

  IscS from Escherichia coli is a cysteine desulfurase that has been shown to be involved in Fe-S cluster formation. The enzyme converts L-cysteine to L-alanine and sulfane sulfur (S(0)) in the form of a cysteine persulfide in its active site. Recently, we reported that IscS can donate sulfur for th ... >> More

  IscS from Escherichia coli is a cysteine desulfurase that has been shown to be involved in Fe-S cluster formation. The enzyme converts L-cysteine to L-alanine and sulfane sulfur (S(0)) in the form of a cysteine persulfide in its active site. Recently, we reported that IscS can donate sulfur for the in vitro biosynthesis of 4-thiouridine (s(4)U), a modified nucleotide in tRNA. In addition to IscS, s(4)U synthesis in E. coli also requires the thiamin biosynthetic enzyme ThiI, Mg-ATP, and L-cysteine as the sulfur donor. We now report evidence that the sulfane sulfur generated by IscS is transferred sequentially to ThiI and then to tRNA during the in vitro synthesis of s(4)U. Treatment of ThiI with 5-((2-iodoacetamido)ethyl)-1-aminonapthalene sulfonic acid (I-AEDANS) results in irreversible inhibition, suggesting the presence of a reactive cysteine that is required for binding and/or catalysis. Both ATP and tRNA can protect ThiI from I-AEDANS inhibition. Finally, using gel shift and protease protection assays, we show that ThiI binds to unmodified E. coli tRNA(Phe). Together, these results suggest that ThiI is a recipient of S(0) from IscS and catalyzes the ultimate sulfur transfer step in the biosynthesis of s(4)U. << Less

  J. Biol. Chem. 275:10727-10730(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Substrate specificity for 4-thiouridine modification in Escherichia coli.

  Lauhon C.T., Erwin W.M., Ton G.N.

  The biosynthesis of 4-thiouridine (s4U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from l-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s ... >> More

  The biosynthesis of 4-thiouridine (s4U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from l-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s4U. In this work, we show through deletion analysis of unmodified E. coli tRNA(Phe) that the minimum substrate for s4U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region. The size of the bulged loop must be at least 4 nucleotides and contain the target uridine as the first nucleotide. Replacement of the T loop sequence with a tetraloop in the deletion substrate increases activity and shows that the TpsiC primary sequence is not a recognition element. An unmodified tRNA(Phe) transcript in which the 3'-terminal ACCA sequence is removed to give a blunt terminus has <0.1% activity, although the addition of a single overhanging base essentially restores activity. In addition, reducing the distance of the 3' terminus relative to U8 by as little as 1 bp severely impairs activity. By dissecting a minimal RNA substrate in the T loop region, a two-piece system consisting of a substrate RNA and a "guide" RNA is efficiently modified. Our results indicate that outside of the modified U8, there is no primary sequence requirement for substrate recognition. However, the secondary and tertiary structure restrictions appear sufficient to explain why s4U modification is limited in the cell to tRNA. << Less

  J Biol Chem 279:23022-23029(2004) [PubMed] [EuropePMC]

• Enzymatic thiolation of E. coli sRNA.

  Lipsett M.N., Peterkofsky A.

  Proc Natl Acad Sci U S A 55:1169-1174(1966) [PubMed] [EuropePMC]

• A [3Fe-4S] cluster is required for tRNA thiolation in archaea and eukaryotes.

  Liu Y., Vinyard D.J., Reesbeck M.E., Suzuki T., Manakongtreecheep K., Holland P.L., Brudvig G.W., Soll D.

  The sulfur-containing nucleosides in transfer RNA (tRNAs) are present in all three domains of life; they have critical functions for accurate and efficient translation, such as tRNA structure stabilization and proper codon recognition. The tRNA modification enzymes ThiI (in bacteria and archaea) a ... >> More

  The sulfur-containing nucleosides in transfer RNA (tRNAs) are present in all three domains of life; they have critical functions for accurate and efficient translation, such as tRNA structure stabilization and proper codon recognition. The tRNA modification enzymes ThiI (in bacteria and archaea) and Ncs6 (in archaea and eukaryotic cytosols) catalyze the formation of 4-thiouridine (s⁴U) and 2-thiouridine (s²U), respectively. The ThiI homologs were proposed to transfer sulfur via cysteine persulfide enzyme adducts, whereas the reaction mechanism of Ncs6 remains unknown. Here we show that ThiI from the archaeon <i>Methanococcus maripaludis</i> contains a [3Fe-4S] cluster that is essential for its tRNA thiolation activity. Furthermore, the archaeal and eukaryotic Ncs6 homologs as well as phosphoseryl-tRNA (Sep-tRNA):Cys-tRNA synthase (SepCysS), which catalyzes the Sep-tRNA to Cys-tRNA conversion in methanogens, also possess a [3Fe-4S] cluster similar to the methanogenic archaeal ThiI. These results suggest that the diverse tRNA thiolation processes in archaea and eukaryotic cytosols share a common mechanism dependent on a [3Fe-4S] cluster for sulfur transfer. << Less

  Proc Natl Acad Sci U S A 113:12703-12708(2016) [PubMed] [EuropePMC]