(Oq) Reductase Reveals Parallels Between Halorespiration and Trna Modification

Discovery of epoxyqueuosine (oQ) reductase reveals parallels between halorespiration and tRNA modification

Zachary D. Miles, Reid M. McCarty, Gabriella Molnar, and Vahe Bandarian1

Department of Chemistry and Biochemistry, University of Arizona, 1041 East Lowell Street, Tucson, AZ 85721-0088

Edited by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved March 10, 2011 (received for review December 15, 2010)

Transfer RNA is one of the most richly modified biological mole- This in vitro reconstitution of the 7-deazapurine core was aided cules. Biosynthetic pathways that introduce these modifications by the discovery of the toyocamycin biosynthetic gene cluster in are underexplored, largely because their absence does not lead Streptomyces rimosus, which contains three genes that had been to obvious phenotypes under normal growth conditions. Queuo- previously shown to be required for the biosynthesis of queuosine sine (Q) is a hypermodified base found in the wobble positions in Bacillus subtilis (10–13). The cluster also contained a GTP of tRNA Asp, Asn, His, and Tyr from bacteria to mankind. Using cyclohydrolase I (GCH I) homolog, which hinted at GTP as being liquid chromatography MS methods, we have screened 1,755 sin- the primary metabolic precursor. GCH I catalyzes the conversion gle gene knockouts of Escherichia coli and have identified the key of GTP to 7,8-dihydroneopterin triphosphate (H2NTP) in the final step in the biosynthesis of Q. The protein is homologous to first step in the biosynthesis of both folic acid and biopterin. B12-dependent iron-sulfur proteins involved in halorespiration. GCH I was subsequently shown to be required for the biosynth- The recombinant Bacillus subtilis epoxyqueuosine (oQ) reductase esis of Q in Escherichia coli (14). In vitro reconstitution was catalyzes the conversion of oQ to Q in a synthetic substrate, as well accomplished by the successive actions of GCH I, 6-carboxy- as undermodified RNA isolated from an oQ reductase knockout 5,6,7,8-tetrahydropterin (CPH4) synthase, 7-carboxy-7-deazagua- strain. The activity requires inclusion of a reductant and a redox nine (CDG) synthase, and preQ0 synthetase, demonstrating that mediator. Finally, exogenously supplied cobalamin stimulates the four enzymatic transformations are required to convert GTP activity. This work provides the framework for studies of the bio- to preQ0, a previously identified intermediate in the biosynthesis synthesis of other modified RNA components, where lack of acces- of queuosine (see Fig. 1). At this point, the biosynthetic pathway sible phenotype or obvious gene clustering has impeded discovery. toward toyocamycin diverges from that of queuosine with preQ0 Moreover, discovery of the elusive oQ reductase protein completes being elaborated to toyocamycin in a series of steps that are ana- the biosynthetic pathway of Q. logous to those in purine biosynthesis/salvage. By contrast, the biosynthetic pathway to queuosine entails the NADPH-depen- ∣ ∣ biochemistry queuosine reductive dehalogenation dent conversion of preQ0 to 7-amino-7-deazaguanine (preQ1)by preQ0 reductase (15, 16). PreQ1 base is then exchanged for gua- early 100 modifications have been identified in RNA, many nine in the anticodon loop of tRNA by tRNA:guanine transgly- Nof which are found in tRNA and are common to eukaryotes, cosylase (Tgt) (6, 17, 18) and elaborated to oQ by addition of a bacteria, and archaea (1). Most of these modifications are likely cyclopentanediol epoxide derived from S-adenosyl-L-methionine not essential under normal laboratory conditions, making dis- by oQ synthase (QueA) (7). The penultimate intermediate covery of biosynthetic pathways by phenotypic methods impossi- to queuosine, oQ, was isolated and characterized 23 y ago (19) ble. Queuosine (Q), a hypermodified RNA base containing a and B12 was subsequently implicated in its conversion to Q (20). 7-deazapurine core, is among the more complex RNA modifica- The identity of the enzyme that catalyzes the transformation and tions described to date. Q replaces the guanine in the wobble in vitro reconstitution of oQ reductase activity are reported in this positions of the subset of tRNA molecules with a 5′-GUN-3′ manuscript. sequence in their anticodon loops (His, Asp, Asn, and Tyr). Con- servation of Q in RNA of organisms in nearly all kingdoms of life Results and Discussion (2) suggests that the modification may be of cardinal importance. Screening of the Keio Collection. The availability of a series of iso- A physiological role for Q has eluded discovery partly because of genic variants of E. coli K-12, the Keio collection (21), in which gaps in understanding of the biosynthetic pathway. De novo bio- each of the approximate 4,000 nonessential genes are individually synthesis of Q occurs in bacteria whereas eukaryotes acquire the disrupted by a kanamycin resistance cassette, provided a valuable free base, queuine, from dietary sources (3–5). The bacterial opportunity to mount a search for the elusive oQ reductase. We pathway for biosynthesis of Q has been elucidated up to the identified 1,755 knockout strains carrying deletions in genes of penultimate intermediate, epoxyqueuosine (oQ) (6, 7). However, unknown function, annotated as “y” genes in the National Center the enzyme that catalyzes the final step in the pathway, conver- for Biotechnology Information genome database (see Dataset S1 sion of oQ to Q, had yet to be identified. Because no selectable for a complete list of deletion strains examined in this study), as phenotypes have been demonstrated for the modification, discov- potential candidates for genes that encode oQ reductase. Each ery of the final step in the pathway required a different approach that melded modern analytical methods and emerging tools in Author contributions: Z.D.M., R.M.M., and V.B. designed research; Z.D.M., R.M.M., G.M., bacterial genetics. This methodology has led to successful iden- and V.B. performed research; Z.D.M., R.M.M., and V.B. contributed new reagents/analytic tification of the final step and can be generalized to other RNA tools; Z.D.M., R.M.M., and V.B. analyzed data; and Z.D.M., R.M.M., and V.B. wrote modifications where lack of phenotype has hindered discovery of the paper. the biosynthetic pathway. The authors declare no conflict of interest. Structural parallels between the 7-deazapurine core of queuo- This article is a PNAS Direct Submission. sine and 7-deazapurine-containing antibiotic toyocamycin were 1To whom correspondence should be addressed. E-mail: [email protected]. noted long before the various elements of the biosynthetic path- This article contains supporting information online at www.pnas.org/lookup/suppl/ way leading to either compound was reconstructed in vitro (7–9). doi:10.1073/pnas.1018636108/-/DCSupplemental.

7368–7372 ∣ PNAS ∣ May 3, 2011 ∣ vol. 108 ∣ no. 18 www.pnas.org/cgi/doi/10.1073/pnas.1018636108 Downloaded by guest on September 26, 2021 Fig. 1. Biosynthetic pathway for production of queuosine with the oQ reductase step elucidated in this manuscript boxed. The block arrow denotes a series of steps analogous to purine salvage/repair, which convert the 7-cyano-7-deazaguanine base to toyocamycin.

deletion strain was grown in Lennox broth, RNA was isolated from each culture, digested, and dephosphorylated (22–24). Nucleosides were resolved by reverse phase HPLC and detected by tandem UV-visible spectrophotometry and mass spectrometry. Under these conditions, oQ and Q, which are present in minis- cule amounts, are readily detected and identified. Representative UV-visible and extracted ion chromatograms obtained from wild-type E. coli as processed and described above are shown in Fig. 2A. Queuosine and epoxyqueuosine typically elute at approximately 30–32 min in chromatograms. Extracted ion chromatograms monitoring oQ (m∕z ¼ 426) and Q (m∕z ¼ 410) show that under normal growth conditions both forms are present in RNA (Fig. 2B). However, the intensity of the peak for oQ is typically 0–30% of that of Q. RNA isolated from any strain carrying deletions up to and including Tgt would be pre- dicted to be completely devoid of Q and oQ (6). Indeed, deletion of CPH4 synthase (b2765), CDG synthase (b2777), preQ0 synthethase (b0444), preQ0 reductase (b2794), and Tgt (b0406) lead to disappearance of both Q and oQ from RNA. GCH I is an essential gene for E. coli because it also catalyzes the first step in biosynthesis of folic acid (25, 26), and is therefore not included Fig. 2. Analysis of RNA from Keio collection deletion strains for presence in the Keio collection. Interestingly, although oQ or Q are absent and absence of Q and oQ. A shows a representative UV-visible trace of the RNA samples, highlighting the region where oQ and Q elute. Wild-type in extracted ion chromatograms of RNA from the QueA (b0405) E. coli B m∕z ¼ contain very little oQ when grown in LB. shows extracted ion chro- deletion strain, the precursor to oQ, preQ1 nucleoside ( matograms of wild-type and deletion strains corresponding to each step in 312), is clearly present (see Fig. 1 for structure). the pathway. Deletion of any gene up to and including Tgt leads to complete Liquid chromatography (LC) MS analysis of y genes in the absence of oQ (m∕z ¼ 426)orQ(m∕z ¼ 410). Deletion of queA leads to Keio collection revealed a single gene, yjeS (b4166), which is accumulation of preQ1 nucleoside (m∕z ¼ 312), as would be expected (see required for conversion of oQ to Q. RNA from the ΔyjeS strain Fig. 1). Deletion of queG leads to complete absence of Q and accumulation clearly lacks Q and accumulates oQ, as would be expected if YjeS of oQ. A seven-point data smoothing algorithm was applied to all of the extracted ion chromatograms. catalyzes conversion of oQ to Q in vivo. We observed the identical phenotype with the deletion of tonB (see Fig. S1), which is a peri- plasmic protein that is required for import of cobalamin and Fe Bioinformatic Analysis of oQ Reductase. QueG is annotated to be a (27)—both of which are cofactors for oQ reductase (see below). Fe-S containing protein involved in electron transport. A BLAST

The YjeS protein will hereafter be referred to as QueG or oQ (28, 29) search with the QueG sequence, however, reveals BIOCHEMISTRY reductase. similarity to a class of corrinoid Fe-S containing proteins from

Miles et al. PNAS ∣ May 3, 2011 ∣ vol. 108 ∣ no. 18 ∣ 7369 Downloaded by guest on September 26, 2021 anaerobic halorespiring bacteria. These proteins are found in organisms that grow in biological niches that are heavily contami- nated by halogenated organics in which halogenated compounds serve as terminal electron acceptors (see ref. 30 for a review). Recent genome sequencing of a perchloroethene dechlorinating bacterium, Dehalococcoides ethenogenes, has revealed 17 homologs in this organism alone, which are presumably specialized for breakdown of different halogenated compounds (31). Fig. S2 shows an alignment of several bacterial QueG homologs and the biochemically studied (32) tetrachloroethene reductase from Dehalobacter restrictus highlighting conservation in QueG of the eight Cys residues that comprise the two 4Fe-4S clusters in this and other reductive dehalogenase proteins studied to date. Sequence similarities between QueG and reductive dehalogenases are localized to the C-terminal half of the dehalogenases. The N termini of dehalogenase proteins often contain a region involved in membrane association, which is presumably to prox- imate the protein with the electron transport chain. A cobalamin cofactor is found in all reductive dehalogenase proteins studied to date (30, 32–36), which is intriguing given the long-standing observation from Kersten and coworkers suggesting the involve- ment of cobalamin in the conversion of oQ to Q (20) and the requirement for presence of TonB demonstrated in this manuscript. Intriguingly, we also find conserved D109xH111 (E. coli numbering) motif in the reductase, which we hypothesize, by analogy to cobalamin-dependent methionine synthase (37), may be involved in the base-off binding of the cobalamin cofactor. Such a motif is also found in a subset of reductive dehalogenase proteins (38). Although the motif is not universally conserved in all reductive dehalogenase proteins, spectroscopic evidence suggests base-off binding of the cofactor to several dehalogenases (32, 35).

In Vitro Reconstitution of oQ Reductase Activity. Because of solubility issues relating to the expression of the E. coli YjeS protein, we chose to isolate the B. subtilis protein which is annotated as YhbA (BSU08910) and shares 30% sequence identity with the E. coli protein; all of the key sequence features of the E. coli homolog are conserved in the B. subtilis protein. Heterologous expression of His6-YhbA in E. coli in the presence of Fe and cobalamin at 18 °C led to accumulation of significant quantities of soluble protein. As with the anaerobic reductive dehalogenases, we expect oQ reductase to be oxygen sensitive. Hence the protein was purified and characterized under strictly anaerobic conditions (95–97% N2∕3–5% H2). Fractions from the Ni-affinity column were orange/brown. Attempts at desalting the protein to remove imidazole and NaCl led to loss of the chromophore. Inclusion of glycerol (20% vol∕vol), however, did improve reten- tion. Because the protein was judged to be >85% pure (based on SDS-PAGE) as it eluted from the column, we opted to utilize it without any additional purification steps. We utilized a synthetic substrate to establish QueG activity in vitro. Both Tgt and QueA have been shown to catalyze their respective transformations on a synthetic 17-mer oligoribonu- cleotide stem loop substrate corresponding to nucleotides 28–44 Fig. 3. Activity assays with oQ reductase. Incubation of oQ reductase in the of the E. coli tyrT tRNA (39). To prepare the substrate for the presence of methyl viologen, dithionite, and B12 leads to time-dependent disappearance of the oQ and appearance of Q (A). In the time course, the areas analysis, preQ1 was generated enzymatically from synthetic of the extracted ion chromatograms at m∕z ¼ 426 or 410, corresponding to preQ0 (40), exchanged into the wobble position of the stem loop oQ or Q, respectively, were measured and plotted. The intensity of the peaks with Tgt, and modified to oQ with QueA. Control experiments in at each time point were referenced to the intensities of the unreacted sub- which the substrate was digested to the corresponding nucleo- strate (100% oQ) or full conversion (100% Q). B12 stimulates activity by sides and analyzed by LC-MS show that >85% of the substrate approximately 30%. Control experiments show that methyl viologen and is modified to oQ nucleoside (m∕z ¼ 426), as judged by compar- dithionite are absolutely required as shown in extracted ion chromatograms ison of the oQ peak to that of guanosine. The initial assays with in B. A seven-point data smoothing algorithm was applied to all of the the protein were carried out with all of the components that we extracted ion chromatograms. reasoned might be required for activity. The purified protein catalyzes the conversion of oQ in the synthetic substrate to Q under reaction is complete in approximately 5 min. As shown in Fig. 3B, anaerobic conditions in a time-dependent manner (Fig. 3A), as no turnover is observed when either the enzyme, dithionite, or monitored by disappearance of oQ and appearance of Q. The methyl viologen are removed. Intriguingly, the reactions are

7370 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1018636108 Miles et al. Downloaded by guest on September 26, 2021 stimulated in the presence of cobalamin (see Fig. 3A). The stimu- able to studies aimed at identification of proteins involved in lation is not large (approximately 1.3-fold) but it is reproducibly other RNA modification pathways where physiological function observed, suggesting that approximately 30% of the active pro- of the modification has remained unknown. tein contains Fe-S but no cobalamin. The requirement for dithionite and methyl viologen, however, was established by showing Methods complete absence of Q in the synthetic stem loop substrate even Growth of E. coli Strains and Isolation of Total RNA. Wild-type and E. coli dele- after a longer incubation period (15 h) and in the presence of tion strains from the Keio collection were inoculated into 50 mL Lenox Broth 34 μ ∕ large amounts of enzyme (20-fold higher); the extracted ion chro- (LB) containing g mL kanamycin sulfate, grown for 16 h at 37 °C, and harvested by centrifugation. Total cellular RNA was extracted using a proto- matograms from these studies are identical to controls shown col based on Kingston et al. (24). Each E. coli cell pellet was resuspended in in Fig. 3B. We note that the requirement for a reductant and a 0.6 mL of a denaturation solution containing 4 M guanidine thiocyanate, mediator, such as dithionite and methyl viologen, have also been 25 mM sodium citrate (pH 7.0), 0.5% N-laurylsarcosine, and 0.1 M β-mercap- demonstrated for all reductive dehalogenase proteins studied to toethanol. The following were added to the resuspended cells in the order date (30). The cellular reductant has never been identified. listed: 0.1 mL of 2 M sodium acetate (pH 4.0), 1 mL phenol, and 200 μLof To further confirm the oQ reductase activity in the context of bromochloropropane. Each was mixed in by vortexing upon addition. The a representative biological sample, the RNA purified from a mixture was placed on ice for 15 min and centrifuged at 15;000 × g for Δ 15 min to separate aqueous and organic phases. The aqueous layer was queG knockout of E. coli, which contains only oQ (see Fig. 2B), −20 was treated with purified oQ reductase protein. The nucleosides removed, combined with 1 vol of isopropanol, chilled at °C for 30 min, and centrifuged for 10 min at 21;000 × g to pellet the precipitated RNA. The obtained after digestion and dephosphorylation of the RNA were RNA pellet was dissolved in 50 μL of RNase free water and further purified as analyzed by LC-MS. As with the synthetic substrate, QueG cat- follows using RNeasy RNA purification kit (Qiagen) according to manufac- alyzes the conversion of oQ (presumably associated with Asp, turer’s protocol. Asn, His, and Tyr tRNA) to Q (see Fig. S3). These results confirm the role of oQ reductase and bacterial homologs in conversion of Digestion and Dephosphorylation of Isolated RNA. Total cellular RNA purified oQ to Q in vivo. as described above was hydrolyzed to its constitutive nucleotides and depho- The role of B12 in the reaction catalyzed by oQ reductase sphorylated using a procedure that yields samples that are suitable for LC-MS remains to be established. However, one may envision two poten- analysis in positive ion mode (22). Each RNA sample (50 μL in water) obtained μ tial roles for the cofactor, as for example proposed for reductive as described above was combined with 5 L of 0.1 M ammonium acetate (pH 5.3) and 1 μL of nuclease P1 (1.4 U∕μL) (Sigma) and incubated for 6 h dehalogenation of perchloroethylene and trichloroethylene (41). at 45 °C. Next, 5 μL of 1 M ammonium bicarbonate and 3 μL of phosphodies- First, cobalamin may be intimately involved in the chemical trans- terase I (2 U∕μL) (Sigma) were added and the mixture was incubated at 37 °C formation either as a covalent or direct electron transfer catalyst for 2 h. The reaction was subsequently combined with 0.1 mL of a solution (41). For example, cobalamin in the +1 oxidation state may containing 10 mM Tris•HCl (pH 7.5) and 10 mM MgCl2 and 3 μL of bacterial attack the C-O bond of the epoxide leading to ring opening. Het- alkaline phosphatase (150 U∕μL) (Invitrogen) and incubated at 37 °C for 2 h. erolytic cleavage of the bond followed by elimination of water The total reaction was filtered through a YM-10 centrifugal filter (Pall Life would lead to formation of Q and oxidation (by two electrons) Sciences) at 12;000 × g for 10 min to remove protein and the flow-through of the cobalamin to the cob(III)alamin state. In this scenario, the containing the nucleosides was analyzed as described below. Fe-S clusters serve to deliver reducing electrons to the cobalamin cofactor to regenerate the nucleophilic +1 state (see Fig. S4). A LC-MS Analysis of RNA Hydrolysates. The presence or absence of oQ and queuosine from cellular RNA was determined by LC-MS of the dephosphory- second mechanistic paradigm would utilize the cobalamin cofac- lated RNA nucleotides. The procedure described here is based on analytical tor as a conduit through which electrons are transferred to the methods reported by Pomerantz and McCloskey (23) for detection and Fe-S cluster(s), where conversion of oQ to Q takes place by an identification of modified bases in RNA. An aliquot (30 μL) of RNA nucleo- unknown mechanism. Studies to differentiate between these and sides prepared as described above was injected onto a 4.6 × 250-mm other mechanistic possibilities could be undertaken with recom- Eclipse XDB-C18 column (Agilent) which had been preequilibrated in binant oQ reductase using site-directed mutagenesis experiments 19.3% ðwt∕volÞ ammonium acetate adjusted to pH 6.0 with glacial acetic acid in which the Fe-S cluster(s) or the cobalamin binding determi- (solution A) and run at 0.3 mL∕ min. Nucleosides were eluted from the col- nants are removed. Moreover, given the protocols that permit umn with a complex gradient from solution A and a solution composed of 40% acetonitrile and 60% H2O (solution B). The separation program was as isolation and analysis of cellular RNA for oQ and Q, it should follows: 0–3 min, 0% solution B; 3.0–4.4 min 0–0.2% solution B; 4.4–5.8 min, be possible to obtain RNA from cells grown under various 0.2–0.8% solution B; 5.8–7.2 min, 0.8–1.8% solution B; 7.2–8.6 min, 1.8–3.2% conditions, to probe the role of the cofactors in vivo with chro- solution B; 8.6–10 min, 3.2–5% solution B; 10–25 min, 5–25% solution B; mosomal copies of the oQ reductase gene where Fe-S or coba- 25–40 min, 25–50% solution B; 40–49 min, 50–75% solution B; 49–52 min, lamin binding determinants have been deleted. Moreover, 75% solution B; 52–60 min, 75–100% solution B. The elution was monitored examination of deletion strains may also provide insights into by UV-visible and MS detection. UV-visible spectra (220–500 nm) were the in vivo reductant for the oQ reductase reaction. recorded on a ThermoFinnigan Surveyor photodiode array detector. MS was The TonB requirement for the modification may be important carried out with an in-line electrospray (ES) ionization-equipped LCQ Ther- moFinnigan Deca XP mass spectrometer, which was operated in the positive physiologically. In addition to B12 and iron, TonB has been impli- ion mode. The m∕z range of 90–500 atomic mass unit was scanned and the cated in the import of colicins into E. coli (42). Colicins are ES was set at a 7 V ionization energy and a 200 °C ion source temperature. bacterial toxins, which upon uptake into a susceptible host, exert their lethal effects. ColE5, for example, is a ribonuclease that Cloning of B. subtilis QueG. The open reading frame encoding B. subtilis QueG specifically cleaves on the 3′ side of Q-containing tRNAs in E. coli (yhbA) was amplified from B. subtilis chromosomal DNA by PCR using (43). Many colicins share the same BtuB–TonBsystem that is used the following primers: 5′- CATATGAACGTTTATCAGCTCAAAGAAGAATTAATT- for import of iron and B12. Although a role for TonB in mediating GAATACGCG -3′ (forward) and 5′- CTCGAGTCATCAGGACAGGCCTTGTTTAGT- ColE5 uptake has not been established, at present one cannot rule CATGCCTGAAG -3′ (reverse) at an annealing temperature of 53 °C. The resulting PCR product was subcloned into pGEM-T Easy vector (Promega) out a link between TonB and import of ColE5, B12, and iron. prior to being excised by NdeI and XhoI and being cloned into similarly digested pET28a (Novagen) to yield pZM002 for expression of His6- Summary. These studies complete the biosynthetic pathway for tagged QueG. queuosine and pave the way for probing the physiological role of queuosine, which, based on its ubiquity, we suspect serves Expression of B. subtilis oQ Reductase. E. coli HMS-174 (DE3) containing a central role in biology. Moreover, the methodology used in this pZM002 (for expression of QueG) and pBD1282 (from Dennis Dean, Virginia study to identify a B12-dependent protein involved in the bio- Tech, Blacksburg, VA, through Squire Booker, Pennsylvania State University, BIOCHEMISTRY synthesis of queuosine is relatively simple and generally applic- University Park, PA), which contains the isc operon of Azotobacter vinelandii,

Miles et al. PNAS ∣ May 3, 2011 ∣ vol. 108 ∣ no. 18 ∣ 7371 Downloaded by guest on September 26, 2021 were grown in 6 L of LB containing 34 μg∕mL kanamycin and 100 μg∕mL the binchoninic acid method using bovine serum albumin (Thermo Scientific) ampicillin at 37 °C to an OD600 of approximately 0.3, at which point solid ara- as a standard. binose was added to a final concentration of 0.05% ðwt∕volÞ to induce tran- scription of the genes in pBD1282. The cells were grown further to an OD600 Assay for Conversion of oQ to Q by E. coli QueG. The conversion of oQ to of approximately 0.5, at which point ferric chloride (final concentration of queuoseine by QueG was carried out using either a 17-mer oQ-containing 50 μM) was added and expression of QueG was induced by addition of IPTG RNA stem loop, prepared as described in SI Text, or total RNA from the ΔqueG (0.1 mM). Hydroxocobalamin (acetate salt, 10 μM final concentration) was strain of E. coli. The assays contained 12 mM Hepes•NaOH (pH 7.2), 1.5 mM added as well. Cells were grown for 8 h at 18 °C, harvested by centrifugation hydoxocobalamin (acetate salt), 1.5 mM methyl viologen, 20 mM sodium (5;000 × g), frozen in N2, and stored at −80 °C. dithionite, 4 μL of either the synthetically modified 17-mer stem D loop (35 μM) containing epoxyqueuosine or purified total RNA (3.2 mg∕mL) from Purification of E. coli QueG. Purification of QueG was carried out in a Coy the ΔqueG strain of E. coli,and6μM QueG protein. The final solutions also anaerobic chamber (approximately 95–97% N2, 3–5% H2). Cells (approxi- contained 1.3 mM potassium phosphate (pH 7.2), 33.3 mM sodium chloride, mately 3 g) were suspended in buffer containing 20 mM potassium phos- 20 mM imidizole (Sigma), and 1% ðvol∕volÞ glycerol carried over from the phate (pH 7.2), 0.5 M NaCl, 5 mM imidazole, 20% glycerol, and 1 mM QueG stock solution. The reactions were initiated by addition of QueG PMSF and sonified using a Branson digital sonifier (45% amplitude). The and quenched by addition of 0.25 mL QIAzol reagent (which contains phe- lysate was centrifuged for 20 min at 14;500 × g and 4 °C to remove cellular nol). RNA was purified from the reaction mixtures using the protocol debris. The cleared lysate was loaded onto a 1 mL HisTrapHP column (GE described above for the cleanup of cellular RNA with the RNAeasy kit, Healthcare), which had been charged with NiSO4 and equilibrated with a digested, dephosphorylated, and analyzed by LC-MS as described. solution containing 20 mM potassium phosphate (pH 7.2), 0.5 M NaCl, 5 mM imidazole, and 20% glycerol (buffer A). The column was rinsed with ACKNOWLEDGMENTS. V.B. is grateful for National Institutes of Health (NIH) 15 mL of buffer A. The column was washed with 2 mL of buffer A containing (GM72623) and Burroughs Wellcome Fund for funding various aspects of 0.1 M imidazole to remove nonspecifically bound proteins. Epoxyqueuosine the work. R.M.M. acknowledges support from Biological Chemistry Training reductase was eluted with 6 mL of buffer A containing 300 mM imidazole. grant from NIH (T32 GM008804). Z.D.M. is supported by Integrative Graduate Fractions containing the desired protein were identified using SDS-PAGE, and Education and Research Traineeship Program in Comparative Genomics stored at −80 °C in small aliquots. Protein concentration was determined by (National Science Foundation 0654435).

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