Biosynthesis of Threonylcarbamoyl Adenosine (t⁶A), a Universal tRNA Nucleoside

Background: The modified nucleoside t⁶A is important for tRNA function.

Results: The proteins YrdC/YgjD/YeaZ/YjeE are necessary and sufficient for the biosynthesis of t⁶A in bacteria.

Conclusion: Only the universal protein families YrdC and YgjD are conserved in the biosynthesis of t⁶A among all organisms.

Significance: Elucidating the enzymes responsible for t⁶A biosynthesis, a universal modification of tRNA, is central to understanding its physiological role.

Abstract

The anticodon stem-loop (ASL) of transfer RNAs (tRNAs) drives decoding by interacting directly with the mRNA through codon/anticodon pairing. Chemically complex nucleoside modifications found in the ASL at positions 34 or 37 are known to be required for accurate decoding. Although over 100 distinct modifications have been structurally characterized in tRNAs, only a few are universally conserved, among them threonylcarbamoyl adenosine (t⁶A), found at position 37 in the anticodon loop of a subset of tRNA. Structural studies predict an important role for t⁶A in translational fidelity, and in vivo work supports this prediction. Although pioneering work in the 1970s identified the fundamental substrates for t⁶A biosynthesis, the enzymes responsible for its biosynthesis have remained an enigma. We report here the discovery that in bacteria four proteins (YgjD, YrdC, YjeE, and YeaZ) are both necessary and sufficient for t⁶A biosynthesis in vitro. Notably, YrdC and YgjD are members of universally conserved families that were ranked among the top 10 proteins of unknown function in need of functional characterization, while YeaZ and YjeE are specific to bacteria. This latter observation, coupled with the essentiality of all four proteins in bacteria, establishes this pathway as a compelling new target for antimicrobial development.

Introduction

Nucleoside modification is an intrinsic feature in the maturation of many RNA species (1), and is especially prevalent in transfer RNA (tRNA), where over 100 modified nucleosides have been structurally characterized (2). Particularly striking is the presence of structurally complex modified nucleosides in and around the anticodon, where they play critical roles in translation by maintaining the accuracy of decoding (3). Threonylcarbamoyl adenosine (t⁶A)³ (4), a modified nucleoside located adjacent to the anticodon at position 37 in tRNAs responsible for decoding ANN codons (Fig. 1), is an example of this phenomenon. Structural studies predict an important role for t⁶A in translational fidelity by preventing the formation of a U33-A37 across-the-loop base pairing interaction, as well as allowing cross-strand stacking of A₃₈ and t⁶A₃₇ with the first position of the codon (5–8). In vivo work is consistent with this prediction as yeast mutants devoid of t⁶A exhibit translation related phenotypes such as initiation and frame maintenance defects and increased nonsense suppression (9–11).

FIGURE 1.

The secondary structure of E. coli tRNA^Lys(UUU) (a) and the chemical structure of t⁶A (b).

Elucidation of the biochemical pathways responsible for RNA modifications has been an especially challenging problem (12) perhaps nowhere as striking as for t⁶A. Despite the presence of t⁶A in all domains of life, and the discovery over 30 years ago that t⁶A biosynthesis required ATP, threonine, and bicarbonate (13–17), the biosynthetic enzyme or enzymes have remained undiscovered. Recently, comparative genomic and genetic studies linked the YrdC/Sua5 (18) and YgjD/Kae1/Qri7 (10, 19) families to t⁶A biosynthesis. Notably, both families are universally conserved and were ranked among the top 10 proteins of unknown function in need of functional characterization (20). However, attempts to reconstitute the pathway in vitro with purified recombinant proteins were unsuccessful (10, 19), suggesting that other proteins might be involved.

Clues to the identity of the remaining t⁶A biosynthetic enzymes came from two observations: first, YgjD was shown to form an essential interaction network with the YeaZ (a paralog of YgjD) and YjeE proteins (21). Indeed, the essentiality phenotype of all three corresponding deletion mutants is suppressed by overexpressing the response regulator RstA, and YeaZ was shown to bind both YgjD and YjeE (21). Second, complementation of the essentiality phenotype of the Escherichia coli ygjD deletion mutant by the Bacillus subtilis ygjD gene required co-expression with the B. subtilis yeaZ gene (10), suggesting that the YgjD/YeaZ physical interaction is specific and relevant to t⁶A biosynthesis in vivo. These observations together with the strong physical clustering of ygjD, yeaZ, and yjeE genes (10, 21) led us to investigate whether YeaZ and YjeE might also be involved in t⁶A biosynthesis in bacteria.

EXPERIMENTAL PROCEDURES

General

Oligonucleotides were purchased from Integrated DNA Technologies, Inc. Protein concentrations were based on the Bradford dye-binding procedure (22). DNA sequencing was carried out by the OHSU core sequencing laboratory. We thank Christopher Lima (Sloan-Kettering Institute) for the generous gift of the plasmid pSMT3.

Strains and Growth Conditions

Cells were grown in LB at 37 °C unless otherwise noted. When necessary, LB was supplemented with kanamycin (50 μg/ml), ampicillin (100 μg/ml) isopropyl-β-d-thiogalactopyranoside (IPTG, 1 mm), and ZnCl₂ (0.5 μm). Plasmids were transformed into E. coli NovaBlue cells for isolation of plasmid DNA, or E. coli BL21(DE3) for production and isolation of recombinant proteins.

Construction of Expression Constructs for YeaZ (pCDII29) and YjeE (pCDII30)

PCR-based cloning of the E. coli yeaZ and yjeE genes was carried out using genomic DNA from E. coli K12 and the following primers: YjeE Forward: 5′-GGTATTGAGGGTCGCATGATGAATCGAGTAATTCCG-3′; YjeE Reverse: 5′-AGAGGAGAGTTAGAGCCTTAACCGGCTAAACGCGCCAG-3′; YeaZ Forward: 5′-GGTATTGAGGGTCGCATGCGAATTCTGGCTATCG-3′; YeaZ Reverse: 5′-AGAGGAGAGTTAGAGCCTCATTCTTTGCCCGGAAG-3′. PCR reactions contained 500 ng of genomic DNA (E. coli K12), 200 μm dNTPs, 50 pmol primers, 1X Pfu Ultra buffer (supplied by the manufacturer), and 2.5 units of Pfu Ultra DNA polymerase in a final volume of 50 μl. A three step PCR thermocycling protocol was utilized: 1) 94 °C for 3 min; 2) 30 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 2 min; 3) 72 °C for 3 min. The PCR products were then gel purified (1% agarose), the DNA isolated (Qiagen PCR purification kit) and ligated into a FactorXa/LIC vector (Novagen) as described by the manufacturer. The primary structures of the resulting constructs were confirmed by sequencing.

Overproduction and Purification of Recombinant, Affinity-tagged YrdC, YeaZ, YjeE, and YgjD

Cultures of transformed cell lines containing pBY215.1 (YrdC), pCDII29 (YeaZ), pCDII30 (YjeE), or pCD174 (YgjD) were grown overnight at room temperature until an A₆₀₀ of 2–3 was obtained. Cells were pelleted (2,500 × g, 4 °C) and resuspended in an equal volume of LB. After a 1-h recovery period at 20 °C (YgjD) or 37 °C (all others), IPTG was added to a final concentration of 1.0 mm, and cultures were grown for an additional 4 h, or for YgjD 16 h in the presence of 0.5 μm ZnCl₂. Cells were then pelleted (5000 × g, 15 min, 4 °C) and resuspended to 250 mg/ml in lysis buffer comprised of 100 mm Tris-HCl (pH 8.0), 2.0 mm 2-mercaptoethanol (BME), 1.0 mm phenylmethylsulfonyl flouride (PMSF), 10% glycerol, and 100 mm KCl or 300 mm KCl (ygjD). Lysozyme (0.25 mg/ml) was then added, and the resulting solutions incubated for 30 min, followed by 3 cycles of freeze thaw and the addition of DNase (10 μg/ml). The solutions were centrifuged (15,000 × g, 20 min, 4 °C), and the supernatants applied to prequilibrated Ni-NTA (Qiagen) columns. The columns were washed sequentially with 7 volumes of lysis buffer, 3 volumes of lysis buffer + 20 mm imidazole, 4 volumes of lysis buffer (without PMSF) + 30 mm imidazole, followed by elution with lysis buffer (without PMSF) + 200 mm imidazole. Fractions containing purified protein were concentrated using Millipore^TM Centrifugal Filter units and subjected to dialysis (Slyde-a-lyzer^TM, Pierce) against 100 mm Tris-HCl (pH 8.0) and 100 mm KCl or 300 mm KCl (YgjD) at 4 °C. The fusion proteins were cleaved overnight at room temperature using either Factor Xa (1 μg/mg, YrdC/YeaZ/YjeE) (New England Biolabs^TM) or ULP1 (10 μg/mg, YgjD) according to the manufacturer's (Factor Xa) or published (ULP1) protocols (23), and the solutions then applied to a pre-equilibrated Ni-NTA column. After eluting with 100 mm Tris-HCl (pH 8.0), 300 mm KCl, and 2 mm BME, the protein pools were concentrated (Millipore^TM Centrifugal Filter units), combined with an equal volume of glycerol, and stored at −80 °C for future use.

Construction of tRNA Templates for in Vitro Transcription

The templates for producing tRNA^Lys and tRNA^Gln transcripts were produced via a Klenow extension reaction as described (24) with the following primers: Forward tRNA^Lys: 5′-AATTCCTGCAGTAATACGACTCACTATAGGGGTCGTTAGCTCAGTTGGTAGAGCAG-3′; Reverse tRNA^Lys: 5′-TGGTGGGTCGTGCAGGATTCGAACCTGCGACCAATTGATTAAAAGTCAACTGCTCTACC-3′; Forward tRNA^Gln: 5′-TGCAGTAATACGACTCACTATAAGTCCCGTGGGGTAGTGGTAATCCTGC-3′; Reverse tRNA^Gln: 5′-TGGTAGTCCCGAGCGGAGTCGAACCGCTGTCGCCGGGTCCAAAGCCCAGCAGGATTAC-3′.

RNA Transcription and Purification

Transcription reactions to produce a 17-nucleotide RNA corresponding to the anticodon stem-loop of tRNA^Lys (17-mer RNA) or the full-length E. coli tRNAs were run overnight at 37 °C in 80 mm HEPES (pH 7.5), 2.0 mm spermidine, 24 mm MgCl₂, 2.0 mm NTPs, 300 pmol template, and 2.5 μg of T₇ polymerase. The template for tRNA^Thr production was generated by MvaI digestion of pCDI147 (10). Prior to reactions the templates were heated to 95 °C for 5 min and allowed to anneal slowly by cooling to room temperature over 1 h. The RNA products generated in the transcription reactions were precipitated by the addition of 0.1 volume 8.0 m ammonium acetate, 3 volumes of 100% ethanol, and cooling at −80 °C for 30 min, then pelleted by centrifugation at 15,000 × g for 30 min at 4 °C, and resuspended in 50 mm HEPES (pH 7.5), 2.0 mm EDTA. The solutions were mixed 1:1 with formamide, boiled for 5 min, and snap cooled on ice before being purified via urea-PAGE electrophoresis (20% 17-mer RNA, 10% tRNA). The RNA was extracted from the gel by crush and soak, then precipitated as above, and resuspended in 50 mm HEPES (pH 7.5) 2 mm EDTA.

Radiochemical Incorporation Assays

Reaction assays measuring the incorporation of [¹⁴C]threonine into RNA were carried out in a volume of 50 μl containing 5 μm each YrdC, SUMO-YgjD fusion, YeaZ, and YjeE, 100 mm Tris-HCl (pH 8), 300 mm KCl, 5.0 mm DTT, 10 mm ATP, 20 mm MgCl₂, 1 μm ZnCl₂, 1 mm MnCl₂, 1 μm CaCl₂, and 50 μm 17-mer RNA or full-length tRNA transcript, 50 mm NaHCO_3, and 50 μm l-[¹⁴C]threonine (100,000 DPM/assay). When [¹⁴C]bicarbonate was investigated 100 μm [¹⁴C]NaHCO₃ (200,000 DPM/assay) and 10 mm l-threonine were present. Assays were run for 20 min at 37 °C, and the RNA precipitated with the addition of 0.1 volume 8 m ammonium acetate and 3 volumes of ethanol, followed by cooling at −80 °C for 30 min. The RNA was then collected by filtration through Whatman GF/B filters, and the filters washed with 70% cold ethanol. The dried filters were combined with RPI^TM Econo-Safe counting mixture and counted on a Beckman 6500 liquid scintillation counter.

Enzymatic ATPase Assays

Reaction assays were carried out in a volume of 10 μl containing 5 μm each YrdC, SUMO-YgjD fusion, YeaZ, and YjeE, 100 mm Tris-HCl (pH 8.0), 300 mm KCl, 5.0 mm DTT, 100 μm [α-³²P]ATP (10,000 DPM), 20 mm MgCl₂, 1 μm ZnCl₂, 1 mm MnCl₂, 1 μm CaCl₂, 40 μm full-length tRNA transcript tRNA^Lys, 50 mm NaHCO_3, and 50 μm l-threonine. The reactions were run for 20 min, then 1 μl aliquots of each assay spotted on PEI-cellulose TLC plates (Silicycle) that were prerun with water and allowed to dry prior to being used for assays. The TLC plates were developed in 0.5 m KH₂PO₄ (pH 3.5), and the radioactivity quantified by phosphorimaging using a GE Typhoon Trio +.

Mass Spectrometric Analysis

tRNA^Lys was placed in reaction buffer containing the same components as the radiochemical incorporation assays, without ¹⁴C-labeled substrates, at a concentration of 1.8 μg/ml. This reaction mixture was incubated at 37 °C for 30 min before phenol/chloroform extraction and ethanol precipitation. This precipitate was subjected to subsequent treatment with nuclease P1, snake venom phosphodiesterase, and alkaline phosphatase as previously described (25). This treated reaction mixture was analyzed by LCMS on a Supelco Discovery HS C18 (250 × 2.1 mm, 5 μm) with a mobile phase of 10 mm ammonium acetate (pH 6.0):acetonitrile (linear gradient of 95:5 to 60:40 over 20 min). Mass analysis utilized an LTQ Orbitrap operating in the positive mode; spray voltage 4.00 kV; capillary temp 300 °C; capillary voltage 49; tube lens voltage 100.

Protein Binding Experiments

Proteins were tested pairwise for binding interactions with one protein His₆-tagged and the other untagged. Proteins were present at 20 μm each in a solution of 100 mm Tris-HCl (pH 8.0), 300 mm KCl, and 2 mm BME (Buffer A), in a final volume of 100 μl. After incubating for 20 min at room temperature the solution was added to 1 ml of Ni-NTA (pre-equilibrated with Buffer A) and exhaustively washed with Buffer A (at least 10 volumes). The bound proteins were then eluted from the column with a solution of Buffer A + 200 mm imadizole. Fractions were pooled and concentrated back to 100 μl using 10 kDa centrifugal filter units (Amicon). Samples were then analyzed by SDS-PAGE.

RESULTS

To test the hypothesis that t⁶A biosynthesis in E. coli requires YrdC, YgjD, YeaZ, and YjeE, the yjeE and yeaZ genes from E. coli were expressed as N-terminal His₆-fusion proteins similar to our construct for yrdC expression (10). While recombinant YrdC, YeaZ, and YjeE were all purified in their native form after removal of the fusion tag, recombinant YgjD proved recalcitrant to soluble expression and required expression as an N-terminal His₆-SUMO fusion (10), which was retained to avoid subsequent precipitation.

The in vitro formation of t⁶A was first probed using radiochemical based assays in which the incorporation of [¹⁴C]threonine or [¹⁴C]bicarbonate into an RNA substrate was measured by liquid scintillation counting after ethanol precipitation of the RNA and collection on glass fiber filters (10). Several RNAs were employed as potential substrates, including unfractionated yeast tRNA from a Δsua5 mutant (18), unmodified E. coli tRNA^Thr and tRNA^Lys transcripts produced through in vitro transcription (both naturally t⁶A containing tRNA), and an unmodified stem-loop RNA corresponding to the ASL of E. coli tRNA^Lys, which has recently been shown to bind YrdC (26). We also tested unfractionated wild-type yeast tRNA which is fully modified with t⁶A, and an in vitro transcribed tRNA^Gln from Methanothermobacter thermautotrophicus, a tRNA that does not naturally contain t⁶A.

Robust incorporation of radioactivity was observed in assays containing [¹⁴C]threonine or [¹⁴C]bicarbonate and all four proteins when the tRNA^Thr and tRNA^Lys transcripts were used, as well as with unfractionated yeast tRNA from the sua5Δ deletion mutant (Fig. 2a), but not when any of the other RNAs were employed. In assays in which one of the proteins or ATP was omitted, no incorporation of radioactivity was observed (Fig. 2b). The observation of specific radiochemical incorporation when either ¹⁴C-labeled threonine or bicarbonate is used, and the failure to observe the incorporation of radioactivity when unfractionated wild-type yeast tRNA or tRNA^Gln were used in the assays, is consistent with the formation of t⁶A, not the nonspecific precipitation of the labeled substrate. Furthermore, the failure to observe incorporation of radioactivity when any one of the four proteins is omitted clearly demonstrates that all four are necessary for t⁶A biosynthesis. The higher activity observed with the purified tRNA^Thr and tRNA^Lys transcripts likely reflect the higher concentration of substrate tRNA in these samples compared with unfractionated tRNA from the Δsua5 mutant. While the failure of the ASL of tRNA^Lys to serve as a substrate is surprising in the context of its established binding to YrdC (26), it is consistent with prior in vivo studies using Xenopus oocytes in which full-length tRNA in its native structure was shown to be necessary for t⁶A formation (27).

FIGURE 2.

Enzyme assays of t⁶A formation. Assays were carried out as described under “Experimental Procedures” with the components indicated. Proteins were present at 5 μm each, with either [¹⁴C]threonine (dark bars, left axis) or [¹⁴C]bicarbonate (light bars, right axis) as the labeled substrate. a, assays with various RNA substrates. tRNA^Thr, tRNA^Lys, tRNA^Gln, and 17-mer indicate in vitro transcribed RNA, ΔSua5 refers to unfractionated tRNA isolated from a Δsua5 yeast strain (17), and WT indicates unfractionated tRNA isolated from wild-type yeast. b, assays with the indicated proteins or ATP absent from the reaction. c, HPLC chromatogram of nucleosides generated from the digestion and dephosphorylation of tRNA^Lys before (bottom trace) and after (top trace) a reaction assay containing YrdC, YgjD, YeaZ, and YjeE. d, partial mass spectrum of compound eluting at ∼9.9 min in the chromatogram.

As a complement to the radiochemical data, and to unambiguously confirm the formation of t⁶A, we carried out in vitro reactions with the tRNA^Lys transcript and unlabeled substrates, digested the modified tRNA to the free mononucleosides (28), and subjected the reaction to analysis by LC-MS (Fig. 2c). A new peak with the retention time of authentic t⁶A is present in the chromatogram of digested tRNA from the reaction assay that is not present in the control sample of unreacted tRNA, and MS analysis of this peak shows it to possess a parent ion with an m/z of 413.14, consistent with protonated authentic t⁶A, as well as a prominent daughter ion at an m/z of 281.05 corresponding to the protonated base.

Having confirmed the in vitro biosynthesis of t⁶A, we investigated the specific role of the four proteins in this process. Conversion of bicarbonate to the urea moiety of t⁶A requires two activation steps with ATP to satisfy the energetic requirements of the acyl-transfer chemistry (29). The first is needed to form an activated bicarbonate, presumably carboxyl phosphate or carboxy-AMP (Fig. 3, I), which can then condense with either the α-amine of threonine or N⁶ of A₃₇ to form an N-carboxyl product (Fig. 3, II). Activation of this product by a second ATP, again via transfer of either phosphate or AMP to generate a second acyl-phospho species (Fig. 3, III), leads to the second condensation reaction with the remaining component to give t⁶A. Thus, AMP and/or ADP should be a co-product of the reaction.

FIGURE 3.

Illustration of the potential chemical paths and intermediates in t⁶A biosynthesis.

To elucidate which product of ATP activation is generated in the reaction we employed assays containing [α-³²P]ATP so that the product or products formed could be directly observed by TLC analysis. Remarkably, both AMP and ADP are produced (Fig. 4a, lane 2), suggesting activation by two distinct mechanisms. In assays in which one of the proteins is absent, AMP production is seen only when YrdC is present in the assay (Fig. 4a, lanes 3, 5, 6), while the formation of ADP is unaffected by the absence of YrdC (Fig. 4a, lane 4), but is significantly reduced when YgjD, YeaZ, or YjeE is absent (Fig. 4a, lanes 3, 5, 6). In the absence of threonine or added bicarbonate (bicarbonate is still present due to ambient CO₂) ADP production is largely unaffected (Fig. 4a, lanes 7–8), while AMP formation is eliminated completely in the absence of threonine (Fig. 4a, lanes 7–8). With assays containing all substrates and a single protein (Fig. 4b, lanes 3–6) or pairs of proteins (Fig. 4b, lanes 7–12), AMP production occurs only in the presence of YrdC, where it is comparable to production in a complete assay, while ADP production is significantly decreased in all assays, and eliminated entirely in the assay where YrdC was used alone (Fig. 4b, lane 6). The observation of both AMP and ADP in assays can suggest the presence of contaminating adenylate kinase, however the absence of AMP when threonine is absent but all of the proteins are present (Fig. 4a, lane 8) indicates that AMP/ADP are legitimate co-products of t⁶A biosynthesis.

FIGURE 4.

TLC analysis of assays with various components of the t⁶A synthesis system using [α-³²P]ATP at 100 μm and the E. coli tRNA^Lystranscript. Assay solutions were prepared as described under “Experimental Procedures” with the indicated component present (+) or absent (−). TLC was carried out on PEI-cellulose plates with a mobile phase of 0.5 m KH₂PO₄ (pH 3.5). a, complete assay (lane 2) or deletions of single proteins (lanes 3–6) or substrates (lanes 7–8). Lane 1 contains only [α-³²P]ATP as a standard. The mobility of AMP and ADP were determined via UV-shadowing with unlabeled nucleotides. b, complete assay (lane 2), individual proteins (lanes 3–6), pairs of proteins (lanes 7–12). Lane 1 contains only [α-³²P]ATP as a standard. c, assays in the absence of a tRNA substrate. Complete assay (lane 2), single proteins (lanes 3–6), pairs of proteins (lanes 7–12), or substrates (lanes 17 & 18). Lane 1 contains only [α-³²P]ATP as a standard. The image of this plate was modified by rearranging the order of lanes.

The above observations clearly show that ATP consumption can be uncoupled from RNA modification, and thus might also occur in the absence of tRNA. Indeed this is the case, when assays lacking tRNA are carried out with varying combinations of proteins and remaining substrates, formation of both AMP and ADP is observed (Fig. 4c). Because uncoupled ATP consumption is common in ATP-dependent systems (30–32) and t⁶A formation requires the hydrolysis of two phosphoanhydride bonds, the simplest explanation of the data is that ATP is utilized to generate two types of activated intermediates, an acyl-phosphate and an acyl-adenylate. It is not yet known whether the observed uncoupling is due to partial reactions or hydrolysis, but this issue is currently being investigated.

The observation that AMP is generated only in the presence of YrdC, and is generated with YrdC alone, clearly establishes that YrdC is responsible for AMP formation. ADP production is clearly more complex, as it drops in the absence of any of the remaining three proteins. This is not entirely surprising, as YjeE and an archaeal YgjD homolog (Kae1) have been previously shown to bind ATP and/or possess weak ATPase activity (33, 34), consistent with our data, and YeaZ is a homolog of YjgD, although it lacks the characteristic ATP binding site of other ATPases of the ASKHA superfamily (35).

Three of the proteins that comprise the t⁶A biosynthetic machinery have been shown to interact physically with each other (YeaZ binds both YjeE and YjgD) (21, 36) raising the possibility that t⁶A formation might occur in a complex involving some or all four proteins. To address this possibility we investigated binding interactions in pull-down experiments utilizing the N-terminal His₆ fusion construct in pairwise experiments in which one protein retained the His₆ fusion and the other had been removed. We confirmed the binding of YeaZ to YjeE and YjgD and the lack of binding between YjeE and YgjD (21, 36) (data not shown). Notably, although we observed that YrdC and YjeE do not interact (Fig. 5), YrdC does bind to both YeaZ and YgjD (Fig. 5), consistent with the formation of a complex or complexes to carry out t⁶A biosynthesis.

FIGURE 5.

Protein binding studies. SDS-PAGE analysis of protein pairs, one without the His₆ fusion and the other retaining the His₆ fusion. Proteins were incubated in buffer for 5 min then applied to a small column of Ni²⁺-affinity resin. The column was washed with buffer to completely remove nonspecifically bound protein (verified by SDS-PAGE), then the bound proteins eluted with buffer containing 200 mm imidazole. Odd-numbered lanes contain protein that did not bind to the column (FT, flow-through), even numbered lanes contain protein that was retained on the column (BD, bound). Far-left lane contains molecular weight markers.

DISCUSSION

While the present data do not allow us to conclude unequivocally which proteins are responsible for the specific catalytic activities required for t⁶A formation, our data clearly demonstrate a threonine-dependent ATPase activity for YrdC that generates AMP. These observations are consistent with recent x-ray crystal structures of the Sulfolobus YrdC homolog Sua5 containing bound threonine and AMPPNP (37), and HypF, a hydrogenase maturation protein that possesses both YrdC- and YgjD-like domains (38). In the Sua5 structure the α-N of bound threonine points toward the α-phosphate of AMPPNP, with an intervening space capable of accommodating the carboxyl group of an N-carboxy-threonine substrate. Indeed, N-carboxy-threonine has been successfully docked (10) to an earlier structure of Sua5 containing bound AMP (39). In one of the HypF crystals soaked with ATP and carbamoylphosphate, carbamoyl-AMP is bound to the YrdC-like domain, demonstrating that the YrdC-like domain in HypF is capable of supporting the formation of acyl-adenylates. Taken together, our observations, and the structural data above, are consistent with YrdC catalyzing the adenylation of N-carboxy-threonine (Fig. 3, rxn 3). Given the observation that E. coli YrdC binds selectively to unmodified tRNA (18) and ASL (26) sequences that correspond to t⁶A containing tRNA, YrdC may also catalyze the condensation of N-carboxyAMP-threonine with A₃₇ (Fig. 3, rxn 4). Elucidating the mechanistic details of these steps is currently underway, as is clarifying the roles of the remaining proteins in the formation of carboxyphosphate and its subsequent condensation with threonine.

Although all four proteins (YrdC, YgjD, YeaZ, and YjeE) are strictly required for t⁶A formation in E. coli, this may not be the case in all bacteria. Indeed, unlike YrdC, YgjD, and YeaZ, which are found in all sequenced bacteria to date (10), YjeE is absent in many intracellular and symbiotic bacteria (such as Mycoplasma, Ureaplasma, Wolbachia, Buchnera, and Wigglesworthia species) as well as Cellulomonas flavigena, suggesting that some bacteria can carry out t⁶A synthesis in the absence of YjeE. The absence of both YjeE and YeaZ in Eukarya and Archaea demonstrates that the function or functions of YjeE and YeaZ are carried out by other enzymes in these organisms. Since Kae1 (the YjgD homolog) was first identified as a component of the EKC/KEOPS complex involved in telomere maintenance (40) and transcription of galactose- and pheromone-responsive genes (41), natural candidates for these missing partners are the other members of the KEOPS complex: Bud32, Pcc1, and Cgi121. Indeed yeast bud32, and pcc1 mutants displayed altered t⁶A contents (9, 19). Conservation of KEOPS complex proteins in Archaea is consistent with a primary role in t⁶A biosynthesis (a universal modification), but further work is required to biochemically characterize the role of KEOPS complex subunits in t⁶A synthesis.

While finally resolving the long-standing problem of elucidating the biosynthetic pathway to t⁶A, the discovery of the enzymes necessary for t⁶A biosynthesis also clarifies the function of four conserved and essential enzyme families that have been the focus of extensive investigation, and functional speculation, for over a decade (reviewed in Refs. 10 and 9, 21, 42–45). Indeed, now that a specific role in the biosynthesis of t⁶A has been established for these proteins, the diversity of pleiotropic phenotypes observed in the absence of these proteins or of their homologs in various organisms can be reexamined in light of this defined functionality. Furthermore, the fact that YeaZ and YjeE are specific to bacteria, and that all four proteins are essential in bacteria, establishes t⁶A biosynthesis as a novel target for antimicrobial development. Given their newly established enzymatic role in t⁶A biosynthesis, and the previous choice of TsaA (i.e. threonylcarbamoyl-6-adenosine, or t⁶A) (46) for the methyltransferase involved in the formation of m⁶t⁶A, we propose to rename the YeaZ, YrdC, YgjD, and YjeE enzymes TsaB, TsaC, TsaD, and TsaE, respectively.