Characterization of thiI, a new gene involved in thiazole biosynthesis in Salmonella typhimurium

The biosynthesis of 4-thiouridine (s 4 U) 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 4 U. In this work, we show through deletion analysis of unmodified E. coli tRNA Phe that the minimum substrate for s 4 U 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 T⌿C 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 s 4 U modification is limited in the cell to tRNA.

mentioning

confidence: 77%

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Lauhon

Erwin

Ton

2004

“…The iscS Ϫ strain did not grow in M9/glucose or M63/glycerol minimal media supplemented with leucine (parent strain MC1061 is a leucine auxotroph). Because of earlier reports (12)(13)(14) on the relationship between s 4 U and thiamin biosynthesis and our recent work, we suspected that thiamin was also necessary for growth. However, addition of thiamin to the minimal medium was insufficient for growth.…”

Section: Construction Of the Iscsmentioning

confidence: 99%

“…Interestingly, the nuvC mutant, which lacked s 4 U in its tRNA, also required thiamin for growth in minimal media (12). The thiI gene was recently identified as a requirement for both s 4 U (13) and thiamin synthesis (16) in E. coli and for thiazole biosynthesis in Salmonella typhimurium (14).…”

mentioning

confidence: 99%

The iscS Gene in Escherichia coli Is Required for the Biosynthesis of 4-Thiouridine, Thiamin, and NAD

Lauhon

Kambampati

2000

171

180

IscS, a cysteine desulfurase implicated in the repair of Fe-S clusters, was recently shown to act as a sulfurtransferase in the biosynthesis of 4-thiouridine (s 4 U) in tRNA (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568). In frame deletion of the iscS gene in Escherichia coli results in a mutant strain that lacks s 4 U in its tRNA. Assays of cell-free extracts isolated from the iscS ؊ strain confirm the complete loss of tRNA sulfurtransferase activity. In addition to lacking s 4 U, the iscS ؊ strain requires thiamin and nicotinic acid for growth in minimal media. The thiamin requirement can be relieved by the addition of the thiamin precursor 5-hydroxyethyl-4-methylthiazole, indicating that iscS is required specifically for thiazole biosynthesis. The growth rate of the iscS ؊ strain is half that of the parent strain in rich medium. When the iscS ؊ strain is switched from rich to minimal medium containing thiamin and nicotinate, growth is preceded by a considerable lag period relative to the parent strain. Addition of isoleucine results in a significant reduction in the duration of this lag phase. To examine the thiazole requirement, we have reconstituted the in vitro biosynthesis of ThiS thiocarboxylate, the ultimate sulfur donor in thiazole biosynthesis, and we show that IscS mobilizes sulfur for transfer to the C-terminal carboxylate of ThiS. ThiI, a known factor involved in both thiazole and s 4 U synthesis, stimulates this sulfur transfer step by 7-fold. Extracts from the iscS ؊ strain show significantly reduced activity in the in vitro synthesis of ThiS thiocarboxylate. Transformation of the iscS ؊ strain with an iscS expression plasmid complemented all of the observed phenotypic effects of the deletion mutant. Of the remaining two nifS-like genes in E. coli, neither can complement loss of iscS when each is overexpressed in the iscS ؊ strain. Thus, IscS plays a significant and specific role at the top of a potentially broad sulfur transfer cascade that is required for the biosynthesis of thiamin, NAD, Fe-S clusters, and thionucleosides.

“…By using the same genetic screen, we identified the gene thiI as essential for s 4 U formation (25) shortly after Downs and co-workers (26) identified the same gene as essential for thiamin biosynthesis. We have cloned and overexpressed thiI from E. coli (25).…”

mentioning

confidence: 99%

The Role of the Cysteine Residues of ThiI in the Generation of 4-Thiouridine in tRNA

Mueller

Palenchar²,

Buck

2001

116

135

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.The metabolism of many sulfur-containing biomolecules remains incompletely understood. Among the metabolic pathways requiring further elucidation are those leading to ironsulfur clusters (1-5), biotin (6 -8), molybdopterin (9), lipoic acid (10), thiamin (8, 11), and sulfur-containing bases in RNA (12). The sulfur-containing nucleosides include 4-thiouridine (s 4 U), 1 which is found at position 8 of some bacterial tRNA ( Fig. 1) and serves as a photosensor for near-UV light (12). The s 4 U undergoes a photoactivated 2 ϩ 2 cycloaddition with cytidine 13 when the tRNA is exposed to light of a wavelength similar to the 334 nm absorbance maximum of s 4 U (13-15). The resulting cross-linked tRNA are poor aminoacylation substrates (16), and the accumulation of uncharged tRNA arrests growth by triggering the stringent response (17, 18). Lipsett and co-workers (19,20) investigated the enzymology of s 4 U biosynthesis in Escherichia coli and reported that the overall reaction utilized cysteine as the sulfur donor and required ATP as a substrate. Lipsett and co-workers (20) concluded that two enzymes were required and that one of them also plays a role in thiamin biosynthesis and requires the cofactor PLP for activity (21,22). By using a genetic screen based on the role of s 4 U as a photosensor (18,(22)(23)(24), the genetic loci of two ...