ThiI is an enzyme common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA.Comparison of the ThiI sequence with protein sequences in the data bases revealed that the Escherichia coli enzyme contains a C-terminal extension displaying sequence similarity to the sulfurtransferase rhodanese. Cys-456 of ThiI aligns with the active site cysteine residue of rhodanese that transiently forms a persulfide during catalysis. We investigated the functional importance of this sequence similarity and discovered that, like rhodanese, ThiI catalyzes the transfer of sulfur from thiosulfate to cyanide. Mutation of Cys-456 to alanine impairs this sulfurtransferase activity, and the C456A ThiI is incapable of supporting generation of 4-thiouridine in tRNA both in vitro and in vivo. We therefore conclude that Cys-456 of ThiI is critical for activity and propose that Cys-456 transiently forms a persulfide during catalysis. To accommodate this hypothesis, we propose a general mechanism for sulfur transfer in which the terminal sulfur of the persulfide first acts as a nucleophile and is then transferred as an equivalent of S 2؊ rather than S 0 .

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 ...

Background: Carbon and nitrogen are two signals that influence plant growth and development. It is known that carbon-and nitrogen-signaling pathways influence one another to affect gene expression, but little is known about which genes are regulated by interactions between carbon and nitrogen signaling or the mechanisms by which the different pathways interact.

The Arabidopsis thaliana AtNRT2.1 gene, which encodes a NO3-transporter involved in high-affinity uptake by the roots, is a molecular target of several mechanisms responsible for the regulation of root NO3 -acquisition by the N status of the plant. All levels of AtNRT2.1 expression (promoter activity, transcript level, protein accumulation, transport activity) are coordinately up-regulated in the presence of NO3 -, and repressed by downstream N metabolites. Transgenic plants expressing the GUS reporter gene under the control of upstream sequences of AtNRT2.1 have been studied to identify elements targeted by these two regulatory mechanisms. A 150 bp sequence located upstream of the TATA box that is required for both stimulation by NO3 -and repression by N metabolites of the promoter has been identified. This sequence is able to confer these two regulations to a minimal promoter. Split-root experiments indicate that the stimulation of the chimaeric promoter by NO3 -occurs only at the local level, whereas its repression by N metabolites is mediated by a systemic signal spread to the whole plant. The activity of the cis-acting 150 bp element is also regulated by sucrose supply to the roots, suggesting a possible interaction between N and C signalling within this short region. Accordingly, multiple motifs potentially involved in regulations by N and/or C status are identified within this sequence by bioinformatic approaches. This is the first report of such a cis-acting element in higher plants.

Transcription by RNA polymerase II in trypanosomes deviates from the standard eukaryotic paradigm. Genes are transcribed polycistronically and subsequently cleaved into functional mRNAs, requiring trans splicing of a capped 39-nucleotide leader RNA derived from a short transcript, the spliced leader (SL) RNA. The only identified trypanosome RNA polymerase II promoter is that of the SL RNA gene. We have previously shown that transcription of SL RNA requires divergent trypanosome homologs of RNA polymerase II, TATA binding protein, and the small nuclear RNA (snRNA)-activating protein complex. In other eukaryotes, TFIIB is an additional key component of transcription for both mRNAs and polymerase II-dependent snRNAs. We have identified a divergent homolog of the usually highly conserved basal transcription factor, TFIIB, from the pathogenic parasite Trypanosoma brucei. T. brucei TFIIB (TbTFIIB) interacted directly with the trypanosome TATA binding protein and RNA polymerase II, confirming its identity. Functionally, in vitro transcription studies demonstrated that TbTFIIB is indispensable in SL RNA gene transcription. RNA interference (RNAi) studies corroborated the essential nature of TbTFIIB, as depletion of this protein led to growth arrest of parasites. Furthermore, nuclear extracts prepared from parasites depleted of TbTFIIB, after the induction of RNAi, required recombinant TbTFIIB to support spliced leader transcription. The information gleaned from TbTFIIB studies furthers our understanding of SL RNA gene transcription and the elusive overall transcriptional processes in trypanosomes.Trypanosoma brucei is an important human and domestic animal pathogen that lives in the tissue spaces and bloodstream of host organisms. This flagellated protozoan parasite is responsible for considerable morbidity and mortality in subSaharan Africa; current disease treatment options are limited, costly, and often toxic. Interest in preventing and curing parasite infections is focused on understanding and ultimately exploiting basic genetic mechanisms that are present in T. brucei but are foreign to host metabolism.Trypanosomes have unusual ways of expressing genes: polycistronic pre-mRNAs become stable, translatable mRNAs only after the addition of a 5Ј capped spliced leader (SL) sequence and a 3Ј polyadenylated tail. mRNAs and the SL are transcribed by RNA polymerase II (RNA Pol II), but there are no consensus TATA boxes or other cis-acting elements characteristic of proteinencoding gene promoters in other eukaryotes. The SL RNA gene promoter is the only defined RNA polymerase II-dependent promoter in trypanosomes (12) and has architecture similar to that of metazoan RNA polymerase II-dependent small nuclear RNA (snRNA) genes (16). In the case of trypanosomes, the SL RNA gene promoter contains at least two upstream promoter-proximal elements, termed 13,25).Common to the RNA polymerase II-dependent transcription of eukaryotic mRNA and snRNA genes is the requirement for TATA binding protein (TBP). At many mRNA gene promoters, T...