Structure of a reaction intermediate mimic in t6A biosynthesis bound in the active site of the TsaBD heterodimer from Escherichia coli.

The tRNA modification N6-threonylcarbamoyladenosine (t6A) is universally conserved in all organisms. In bacteria, the biosynthesis of t6A requires four proteins (TsaBCDE) that catalyze the formation of t6A via the unstable intermediate l-threonylcarbamoyl-adenylate (TC-AMP). While the formation and stability of this intermediate has been studied in detail, the mechanism of its transfer to A37 in tRNA is poorly understood. To investigate this step, the structure of the TsaBD heterodimer from Escherichia coli has been solved bound to a stable phosphonate isosteric mimic of TC-AMP. The phosphonate inhibits t6A synthesis in vitro with an IC50 value of 1.3 µM in the presence of millimolar ATP and L-threonine. The inhibitor binds to TsaBD by coordination to the active site Zn atom via an oxygen atom from both the phosphonate and the carboxylate moieties. The bound conformation of the inhibitor suggests that the catalysis exploits a putative oxyanion hole created by a conserved active site loop of TsaD and that the metal essentially serves as a binding scaffold for the intermediate. The phosphonate bound crystal structure should be useful for the rational design of potent, drug-like small molecule inhibitors as mechanistic probes or potentially novel antibiotics.

Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA.

In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification.IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

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

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.

Purification, crystallization and preliminary crystallographic analysis of a 4-thiouridine synthetase-RNA complex.

The sulfurtransferase 4-thiouridine synthetase (ThiI) is involved in the ATP-dependent modification of U8 in tRNA. ThiI from Thermotoga maritima was cloned, overexpressed and purified. A complex comprising ThiI and a truncated tRNA was prepared and crystallized, and X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 102.9, b = 112.8, c = 132.8 Å.

Virulence characteristics of Salmonella following deletion of genes encoding the tRNA modification enzymes GidA and MnmE.

Salmonella is an important foodborne pathogen causing major public health problems throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division (gidA) gene significantly altered Salmonella virulence in both in vitro and in vivo models of infection. In Escherichia coli, GidA and MnmE have been shown to modify several bacterial factors by a post-transcriptional mechanism to modify tRNA. Therefore, we hypothesize that GidA and MnmE complex together to modulate virulence genes in Salmonella using a similar mechanism. To test our hypothesis, and to examine the relative contribution of GidA and MnmE in modulation of Salmonella virulence, we constructed gidA and mnmE single mutants as well as a gidA mnmE double mutant strain of Salmonella. Results from the in vitro data displayed a reduction in growth, motility, intracellular replication, and invasion of T84 intestinal epithelial cells in the mutant strains compared to the wild-type Salmonella strain. The in vivo data showed a significant attenuation of the mutant strains as indicated by the induction of inflammatory cytokines and chemokines, as well as in the severity of histopathological lesions in the liver and spleen, compared to mice infected with the wild-type strain. Also, a significant increase in the LD50 was observed in mice infected with the mutant strains, and mice immunized with the mutants were protected against a lethal dose of wild-type Salmonella. A pull-down assay indicated that Salmonella GidA and MnmE bind together, and HPLC analysis revealed that deletion of gidA and/or mnmE altered Salmonella tRNA modification. Overall, the data suggest MnmE and GidA bind together and use a post-transcriptional mechanism to modify tRNA to regulate Salmonella pathogenesis.

Mechanism of N6-threonylcarbamoyladenonsine (t(6)A) biosynthesis: isolation and characterization of the intermediate threonylcarbamoyl-AMP.

Genetic and biochemical studies have recently implicated four proteins required in bacteria for the biosynthesis of the universal tRNA modified base N6-threonylcarbamoyl adenosine (t(6)A). In this work, t(6)A biosynthesis in Bacillus subtilis has been reconstituted in vitro and found to indeed require the four proteins YwlC (TsaC), YdiB (TsaE), YdiC (TsaB) and YdiE (TsaD). YwlC was found to catalyze the conversion of L-threonine, bicarbonate/CO(2) and ATP to give the intermediate L-threonylcarbamoyl-AMP (TC-AMP) and pyrophosphate as products. TC-AMP was isolated by HPLC and characterized by mass spectrometry and (1)H NMR. NMR analysis showed that TC-AMP decomposes to give AMP and a nearly equimolar mixture of L-threonine and 5-methyl-2-oxazolidinone-4-carboxylate as final products. Under physiological conditions (pH 7.5, 37 °C, 2 mM MgCl(2)), the half-life of TC-AMP was measured to be 3.5 min. Both YwlC (in the presence of pyrophosphatase) and its Escherichia coli homologue YrdC catalyze the formation of TC-AMP while producing only a small molar fraction of AMP. This suggests that CO(2) and not an activated form of bicarbonate is the true substrate for these enzymes. In the presence of pyrophosphate, both enzymes catalyze clean conversion of TC-AMP back to ATP. Purified TC-AMP is efficiently processed to t(6)A by the YdiBCE proteins in the presence of tRNA substrates. This reaction is ATP independent in vitro, despite the known ATPase activity of YdiB. The estimated rate of conversion of TC-AMP by YdiBCE to t(6)A is somewhat lower than the initial rate from L-threonine, bicarbonate and ATP, which together with the stability data, is consistent with previous studies that suggest channeling of this intermediate.

Efficient preparation of 2,4-diaminopyrimidine nucleosides: total synthesis of lysidine and agmatidine.

An efficient route for the synthesis of 2,4-diaminopyrimidine ribosides from cytidine is described consisting of six steps with overall yields >50% and only one chromatographic step. The key amine addition step utilizes LiCl and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to ensure clean conversion to a single tautomeric product. This route has been used to prepare the modified tRNA nucleosides lysidine and agmatidine in quantities suitable for structural characterization.

Substrate specificity for 4-thiouridine modification in Escherichia coli.

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.

Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo.

IscS catalyzes the fragmentation of l-cysteine to l-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys(328) resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys(328) identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s(2)C and ms(2)i(6)A) yet showed wild type levels of Fe-S-independent thionucleosides (s(4)U and mnm(5)s(2)U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase.

Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm(5)se(2)U), is located at the wobble position of the anticodons of tRNA(Lys), tRNA(Glu), and tRNA(1)(Gln). Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm(5)se(2)U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm(5)se(2)U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His(6) tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm(5)se(2)U synthesis is shown to be dependent on 2-selenouridine synthase, SePO(3), and tRNA. Finally, we demonstrate that the conserved Cys(97) (but not Cys(96)) in the rhodanese sequence motif Cys(96)-Cys(97)-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes.

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.

Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli.

Escherichia coli tRNA contains four naturally occurring nucleosides modified with sulfur. Cysteine is the intracellular sulfur source for each of these modified bases. We previously found that the iscS gene, a member of the nifS cysteine desulfurase gene family, is required for 4-thiouridine biosynthesis in E. coli. Since IscS does not bind tRNA, its role is the mobilization and distribution of sulfur to enzymes that catalyze the sulfur insertion steps. In addition to iscS, E. coli contains two other nifS homologs, csdA and csdB, each of which has cysteine desulfurase activity and could potentially donate sulfur for thionucleoside biosynthesis. Double csdA csdB and iscS csdA mutants were prepared or obtained, and all mutants were analyzed for thionucleoside content. It was found that unfractionated tRNA isolated from the iscS mutant strain contained <5% of the level of sulfur found in the parent strain. High-pressure liquid chromatography analysis of tRNA nuclease digests from the mutant strain grown in the presence of [(35)S]cysteine showed that only a small fraction of 2-thiocytidine was present, while the other thionucleosides were absent when cells were isolated during log phase. As expected, digests from the iscS mutant strain contained 6-N-dimethylallyl adenosine (i(6)A) in place of 6-N-dimethylallyl-2-methylthioadenosine and 5-methylaminomethyl uridine (mnm(5)U) instead of 5-methylaminomethyl-2-thiouridine. Prolonged growth of the iscS and iscS csdA mutant strains revealed a gradual increase in levels of 2-thiocytidine and 6-N-dimethylallyl-2-methylthioadenosine with extended incubation (>24 h), while the thiouridines remained absent. This may be due to a residual level of Fe-S cluster biosynthesis in iscS deletion strains. An overall scheme for thionucleoside biosynthesis in E. coli is discussed.