Discovery and Characterization of Trnaile Lysidine Synthetase (Tils)

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FEBS Letters 584 (2010) 272–277

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Review Discovery and characterization of tRNAIle lysidine synthetase (TilS)

Tsutomu Suzuki *, Kenjyo Miyauchi

Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

article info abstract

Article history: In the bacterial decoding system, the AUA codon is deciphered as isoleucine by tRNAIle bearing lysi- Received 12 November 2009 dine (L, 2-lysyl-cytidine) at the wobble position. Lysidine is an essential modification that deter- Revised 21 November 2009 mines both the codon and amino acid specificities of tRNAIle. We identified an enzyme named Accepted 24 November 2009 tRNAIle lysidine synthetase (TilS) that catalyzes lysidine formation by using lysine and ATP as sub- Available online 26 November 2009 strates. Biochemical studies revealed a molecular mechanism of lysidine formation that consists Edited by Manuel Santos of two consecutive reactions involving the adenylated tRNA intermediate. In addition, we deciphered how Escherichia coli TilS specifically discriminates between tRNAIle and the structurally similar tRNAMet, which bears the same anticodon loop. Recent structural studies unveiled tRNA Keywords: tRNA recognition by TilS, and a molecular basis of lysidine formation at atomic resolution. Lysidine Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. TilS AUA codon Wobble modification Anticodon

1. Lysidine plays a critical role in decoding the AUA codon as Ile tRNAIle bearing the CAU anticodon was switched to Met, and this tRNA translates the AUG codon as Met [4]. Therefore, a single L The charging of a tRNA with the correct amino acid is critical for modification converts the codon-specificity from AUG to AUA, the accurate decoding of cognate codons. Post-transcriptional and the amino acid specificity from Met to Ile. modification at the anticodon first (wobble) position participates in the precise decoding of the genetic code, which is mediated by 2. Discorvery of tRNAIle lysidine synthetase (TilS) the codon-anticodon interaction at the ribosomal A-site [1–3]. Lysidine (L, 2-lysyl-cytidine) (Fig. 1A) is a lysine-containing The chemical structure of lysidine implied that lysine is intro- cytidine derivative that occurs at the wobble position of bacterial duced directly into the wobble position of tRNAIle by a putative en- Ile AUA-specific tRNA (Fig. 1B) that has a CAU anticodon when zyme that has a lysine-transfer activity. Since the discovery of Ile unmodified [4,5]. It was shown that Escherichia coli tRNA bearing lysidine in 1988 [5], many researchers have tried to identify an en- the LAU anticodon is charged exclusively with Ile by isoleucyl- zyme (or a gene) responsible for lysidine formation, but no clues tRNA synthetase (IleRS) and recognizes only the AUA codon regarding the biosynthesis of L were reported until 2003. (Fig. 2). According to the chemical structure of L (Figs. 1A and 2), In search of a gene responsible for lysidine formation, we em- 2 conjugation of the lysine moiety at the C position of cytidine in- ployed a genome-wide screen of uncharacterized genes in E. coli 3 duces a tautomeric conversion, with protonation of the N position combined with a comparative genomics approach named ribonu- 4 and imino group formation at the C position. The proton donor– cleome analysis [6–8]. The gene encoding tRNAIle bearing the acceptor pattern of C in hydrogen bonding is completely altered CAT anticodon had been identified in all of the complete genomic to be like that of U by this modification, enabling L to base pair sequences of eubacteria reported at that time when our study com- with A but not with G (Fig. 2). The CAU anticodon also occurs in menced [9], which indicated that L is a universal post-transcrip- Met the tRNA , which is specific to the AUG codon. As the methio- tional modification of eubacterial tRNAIle. Thus, we sought to nyl-tRNA synthetase (MetRS) recognizes the CAU anticodon of identify a gene responsible for lysidine formation by using the Met Ile tRNA , the unmodified tRNA with the CAU anticodon can be Clusters of Orthologous Groups (COGs) of Proteins database [10]. charged with Met instead of Ile (Fig. 2). Indeed, when L was re- A phylogenetic pattern search to identify genes commonly found placed with C using a microsurgery technique, the identity of in E. coli, Bacillus subtilis, and Mycoplasma genitalium resulted in the retrieval of 373 COGs, 48 of which encode hypothetical pro-

* Corresponding author. Fax: +81 3 5841 0550. teins or are uncharacterized genes with an unknown function. As E-mail address: [email protected] (T. Suzuki). the gene responsible for lysidine formation is considered to be

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.11.085 T. Suzuki, K. Miyauchi / FEBS Letters 584 (2010) 272–277 273

ing a partial L modification isolated from the tilS ts strain, and found A B A C that the precursor form of L was unmodified C. This indicated that NH C lysidine formation is a single step reaction that is catalyzed by the A TilS protein. CO2H N G C Ile2 G C To examine whether lysidine formation on tRNA is essential CH(CH2)4HN N C G for the decoding of the AUA codon in the cell, we constructed a re- G NH2 C porter plasmid carrying the mutant b-galactosidase gene (lacZ) HO C G O C G that bears two pairs of tandem AUA codons in its N-terminal re- U A U G 4 A gion. This lacZ reporter was introduced into a tilS-deficient strain s U C G ACC G A A A in which genomic tilS had been disrupted and tilS was rescued in HO OH U C U CG Gm GUGC G T Ψ C trans by another plasmid with a temperature-sensitive (ts) origin G C3 G A G C acp U of replication. When the cultivation temperature was raised to D D A A m7G G U U G the nonpermissive temperature of 42 °C, the rescue plasmid G C harboring tilS gradually disappeared due to its ts replicon. Concur- C G G C rently, the b-gal activity from the reporter plasmid was apparently A Ψ decreased due to its N-terminal tandem AUA codons. These results C A demonstrated that lysidine formation on tRNAIle2 is essential for U t6A AUA decoding in the cell [6]. L U 34 A

Fig. 1. Chemical structure of lysidine and AUA codon-specific tRNAIle2. (A) Chemical 3. Enzymatic formation of lysidine catalyzed by TilS structure of lysidine (L) and 2-lysilcytidine (k2C). (B) Secondary structure of E. coli Ile2 AUA-specific tRNA with the following modified nucleosides: lysidine (L), 4- Sequence alignment of the tilS homologs from various bacteria thiouridine (s4U), 20-O-methylguanosine (Gm), N6-threonylcarbamoyladenosine (t6A), pseudouridine (W), 7-methylguanosine (m7G), 3-(3-amino-3-carboxypro- revealed the presence of a highly conserved N-terminal and a pyl)uridine (acp3U) and 5-methyluridine (T). non-conserved C-terminal region. The N-terminal region bears a highly conserved SGGXDS sequence that was predicted to be a PP-loop motif, which is a common motif used for ATP binding in essential, we selected five candidate COGs from this group, based the ATP pyrophosphatase (PPi synthetase) family [11]. This struc- on a gene essentiality analysis. Among these candidates, we iden- tural feature of TilS implied that lysidine formation is an ATP- tified COG0037, which includes B. subtilis yacA and E. coli mesJ, as dependent reaction. being responsible for lysidine formation [6]. We then renamed this To reconstitute lysidine formation in vitro, E. coli TilS was Ile COG tRNA lysidine synthetase (tilS). Mass spectrometric analyses recombinantly expressed and purified. A gel mobility-shift experi- of the total nucleosides of tRNAs obtained from the cells in which ment showed that TilS specifically interacts with the isolated E. coli tilS was partially inactivated revealed that the level of lysidine was tRNAIle2. The transcribed E. coli tRNAIle or native tRNAIle with par- Ile specifically decreased. In addition, we analyzed E. coli tRNA bear- tial L modification isolated from the tilSts strain (50% of all the

Met Ile

CAU tRNAIle-lysidine synthetase L AU (TilS) AUG AUA

C34 H G L34 H H A N H O N N NH N OHO OH O N H N N N H N N + N N N N O O O O O O + O NH NH NH O 3 O H COO- O OH O OH

Fig. 2. Lysidine converts both the codon and amino acid specificities of tRNAIle. The precursor tRNA bearing C34 can be aminoacylated with methionine and decode the AUG codon. After being modified by TilS, tRNA bearing lysidine gains isoleucine-accepting activity and AUA-codon specificity. Thus, the conversion of both codon and amino acid specificities is governed by this single modification. Chemical structures of base pairing between C34 or L34 and G or A, respectively, at the third codon position is boxed. 274 T. Suzuki, K. Miyauchi / FEBS Letters 584 (2010) 272–277 tRNAIle molecules) was employed as the substrate for lysidine for- First, TilS activates the C-2 position of C34 by forming an adenylate mation. In the presence of ATP, TilS catalyzes lysine incorporation intermediate (Fig. 3). Second, nucleophilic attack of the C-2 posi- into tRNAIle. Mass spectrometric analysis revealed that lysidine tion of the intermediate by the e-amino group of lysine completes was successfully reconstituted in vitro [6]. Based on the kinetic the reaction (Fig. 3). This reaction mechanism is similar to that em- parameters, the transcribed E. coli tRNAIle2 was shown to be a good ployed by the other enzymes in the ATP pyrophosphatase family, substrate for lysidine formation in vitro, showing that the other such as MnmA [13] and GMP synthetase [14]. modifications of tRNAIle2 are not required for its recognition by TilS. 4. Molecular recognition of tRNAIle2 by TilS We then investigated the chemical reaction of lysidine formation in detail. The highly conserved PP-loop motif of the ATP pyro- Mutation studies of E. coli tRNAIle2 were carried out to identify phosphatase family found in the N-terminal domain of TilS is the sites recognized by TilS [12]. In the anticodon loop (Fig. 4), known to participate in the binding and hydrolysis of the a–b replacement of the second (A35) and third (U36) bases of the anti- phosphate bond of ATP. Indeed, our observation that TilS hydro- codon only mildly decreased the activity for lysidine formation, lyzed ATP and produced PPi and AMP during this reaction demon- showing that the second and third bases of an anticodon are not strated that the a–b phosphate bond of ATP is cleaved by TilS [12]. critical for lysidine formation. Replacement of A37, which is 30- Furthermore, a modification intermediate in lysidine formation adjacent to the anticodon, significantly impaired the activity. could be observed. In the absence of lysine in the reaction mixture, Although A37 is modified to t6A37 in native tRNAIle2, the transcript we detected an adenylated cytidine as the modification intermedi- had a similar kinetic parameter as the native transcript. The bulky Ile ate at the wobble position of tRNA . This intermediate is unstable side chain of t6A37 is not recognized by TilS. In addition, U33, Ile2 even at neutral pH, and AMP can be liberated from the tRNA , which is 50-adjacent to the anticodon and a universally conserved Ile2 indicating that the C-2 carbonyl group of C34 in tRNA is acti- residue in most tRNAs, is also required for lysidine formation. vated in the form of adenylate. These results led us to hypothesize U33 is known to stabilize the characteristic U-turn structure of that lysidine is formed through a sequential two-step reaction. the anticodon loop. A one-nucleotide insertion between U33 and

NH2 NH NH

N N CO2H N

AMP O CH(CH2)4HN O N TilS N TilS N NH2 HO O HO O HO O

ATP PPi Lys AMP HO OH HO OH HO OH

Fig. 3. Two-step reaction of lysidine formation. TilS activates the C2 position of C34 and uses ATP as a substrate to form an adenylate intermediate. Then, the e amino group of lysine attacks this position to release AMP and complete the reaction.

tRNAIle2 tRNAMet A A C C C C A A G C G C G C G C C G C G 4 C G 69 4 U A 69 5 C G 68 5 A U 68 C G C G A G A U U A G C U U C A CG C U U C CG C A G A A U G A G U A U A G C U CG GUGC G C C U CG AAGC G C U U G U U G C 44C G A G C 27 43 U G G A G C U U U A A U A A27 43 G G U GG U U C G GG G C A U C G U A G C 30 C G 40 A U A U C A C A U A U A 34C A U 34C A U

L ac4C

Fig. 4. Recognition and discrimination between two tRNAs with CAU anticodons by TilS and TmcA. Schematic depiction of tRNA recognition by TilS in comparison with that by TmcA. TilS strongly recognizes two consecutive base pairs, C4-G69 and C5-G68, in the acceptor stem of tRNAIle2 during lysidine formation, whereas U4-A69 and A5-U68 in tRNAMet act as negative determinants for TilS recognition. In contrast, TmcA strongly recognizes C27-G43 in the anticodon stem of tRNAMet. In addition, C30-G40 and G44 in tRNAMet play a supporting role in TmcA recognition. G27U43 in tRNAIle2 acts as a negative determinant for TmcA recognition. T. Suzuki, K. Miyauchi / FEBS Letters 584 (2010) 272–277 275

C34 abolished the lysidylation activity. These results suggest that hairpin and a subsequent helix-turn-helix (HTH) motif (Fig. 5B). the canonical size and structure of the anticodon loop are required It is suggested that the b-hairpin non-discriminatorily stacks from for recognition by TilS [12]. However, the elongator tRNAMet has the top base pair (G1-C72) of the acceptor helix to the CCA end. The the same sequence in the anticodon loop as does tRNAIle2 (Fig. 4). a-helix of the HTH motif, which contains bulkier aliphatic residues, Therefore, TilS cannot discriminate between tRNAIle2 and the struc- Lys404, Lys 405, Lys407 and Arg420, is accommodated in the major turally similar tRNAMet by recognizing the anticodon loop. groove of the acceptor helix near the identity determinants of Ec- Most variations between these structurally similar tRNAs are TilS, which consist of C4-G69 and C5-G68 (Fig. 5B). Although the found in their acceptor stems and anticodon stems (Fig. 4). When resolution of the crystal structure is not high enough to see de- we swapped their acceptor stems, the mutant tRNAIle2 whose tailed molecular interactions, Lys407 and Arg420 were found to acceptor stem was replaced with that of tRNAMet showed no lysi- be located close to the C4-G69 and C5-G68 in the acceptor helix. dylation activity. In contrast, the mutant tRNAMet whose acceptor When the highly conserved Arg420 was mutated to Ala, the lysi- stem was replaced with that of tRNAIle2 became lysidylated [12]. dine-forming activity was significantly decreased. Arg420 seems This result showed that the sequence difference in the acceptor to be one of the critical residues that recognizes the two consecu- stem plays a critical role in the recognition of tRNAIle2 and in the tive C-G pairs as the identity element [20]. A gel-mobility shift exclusion of tRNAMet by TilS. Further analysis revealed that two experiment revealed that the CTD2 domain can bind tRNAIle2 by it- consecutive C-G pairs at the 4th (C4-G69) and 5th (C5-G68) base self, indicating that CTD2 captures the acceptor helix of tRNAIle2 at pairs from the top of the acceptor stem are mainly recognized by the initial binding stage. TilS, while in tRNAMet, the same base pairs in the acceptor stem, In the ligand-free form of EcTilS, the HTH motif in CTD2 forms a U4-A69 and A5-U68, work as negative determinants for TilS-recog- hydrophobic interaction with CTD1 (the SCL domain). This interac- nition (Fig. 4). We next examined the sites on tRNAIle recognized by tion is disrupted upon tRNA binding, causing the outward rotation TilS. We observed a TilS footprint specifically on the anticodon loop of CTD2 by 59.4° relative to CTD1. The released CTD1 interacts with and the 3’ side of the acceptor stem (positions 64–79) [12], which the inner corner of the L-shaped tRNA and the anticodon arm overlaps with the determinants (C4-G69 and C5-G68) that we (Fig. 5A). CTD1 has a hydrophobic pocket consisting of Leu277 identified earlier. and Leu278, which recognizes A38 in the anticodon loop (Fig. 5C). During landing of the anticodon loop on the crater of 5. Negative determinants for TmcA embedded in tRNAIle the NTD, Arg142 of the NTD enters the anticodon loop, and expels A38 by mimicking the base pairing with C32. The hydrophobic The wobble base of E. coli tRNAMet is modified to N4-acetylcyti- pocket of CTD1 captures the expelled A38, enabling the docking dine (ac4C), which is thought to ensure the precise recognition of of CTD1 to tRNA. The conformational change in the anticodon loop the AUG codon by preventing misreading of the near-cognate places C34 at the catalytic center of TilS (Fig. 5C). AUA codon [15]. We identified an enzyme, which we named tRNA- In a previous study, mutation of Leu277 to Pro was detected in Met cytidine acetyltransferase (TmcA), which catalyzes ac4C forma- the temperature-sensitive mutant of E. coli tilS isolated by plasmid tion at the wobble position of tRNAMet by using acetyl-CoA and ATP shuffling [12]. As the recombinant protein of this mutant showed a as substrates [16]. A structural study revealed that TmcA is a reduced activity for lysidine formation with an increased Km value, GCN5-related N-acetyltransferase fused to an RNA helicase driven it was concluded that the hydrophobic pocket for A38 plays a crit- by ATP hydrolysis [17]. Since TmcA does not recognize the struc- ical role in tRNA binding. In addition, the GkTilS mutant in which turally similar tRNAIle, it was thought that negative determinants Arg142 was replaced with Ala showed reduced activity, demon- for TmcA are embedded in tRNAIle. A mutation study revealed that strating the importance of this residue for anticodon loop recogni- TmcA strictly discriminates between tRNAMet and tRNAIle, mainly tion. These observations were also confirmed by biochemical Ile2 by recognizing the C27-G43 pair in the anticodon stem (Fig. 4). evidence showing that tRNA C32U and A38C mutants had a sig- C30-G40 and G44 in tRNAMet were found to be additional determi- nificant reduction in lysidine-forming activity [20]. nants for TmcA recognition (Fig. 4) [16]. In contrast, tRNAIle has The NTD is a globular catalytic domain that contains the PP- G27-U43, which functions as a negative determinant for ac4C for- loop motif for ATP binding (Fig. 5C). In the apo-form of EcTilS, mation. These studies revealed an elaborate mechanism, embed- the active site of the NTD forms a crater on the inner surface of ded in the two structurally similar tRNAs bearing the CAU the molecule, and a narrow tunnel connects this crater to the out- anticodon, which accurately decodes AUA and AUG codons and is er surface of the NTD. The surface of the crater, composed of sev- based on the recognition of and discrimination between these eral arginine residues, confers a positive surface on which the Ile2 two tRNAs by TilS and TmcA. anticodon loop of tRNA can dock [6]. In the TilS–tRNA complex, lysine is considered to travel to the active site through this tunnel. In the structure of AaTilS complexed with lysine and ATP 6. Structural basis for lysidine formation [19], the adenine base of ATP is buried within the hole in the active site, and is recognized by Phe61, Arg113 and Ser32 (Val54, The crystal structures of the ligand-free form of TilS from E. coli Arg107 and Ser26 in GkTilS, Fig. 5B). The b- and c-phosphate of (PDB code: 1NI5) and A. aeolicus (1WY5) [18], and of A. aeolicus TilS ATP are located at the inner entrance of the hole near the side 2+ complexed with AMPPNP (2E21) and with ATP, Mg and L-lysine chain of Arg201 (Arg199 in GkTilS). In the GkTilS–tRNA complex, (2E89) are available [19]. Recently, the crystal structure of Geoba- Asp131, Gln134 and Arg199, which surround the C34 residue, are Ile2 cillus kaustophilus TilS complexed with B. subtilis tRNA was likely to be involved in the adenylation of C34 and in the subse- determined at 3.65 Å resolution (Fig. 5) [20]. TilS consists of an quent formation of lysidine. Mg2+ coordinated with Asp36 and N-terminal catalytic domain (NTD), one or two C-terminal domains Asp137 in AaTilS (Asp30 and Asp131 in GkTilS) is considered to (CTD), and a long a-helical linker connecting the NTD and CTD. Ec- be involved in the adenylation of C34. In the AaTilS structure, TilS and GkTilS are Type-I TilS proteins, which have a CTD1 and the electric density of lysine was visible near the outer entrance CTD2, whereas AaTilS is a Type-II TilS protein, which contains only of the narrow tunnel (Fig. 5C). As this site is far from the catalytic a CTD1. center where C34 and ATP are bound, it is thought to be an initial In the GkTilS–tRNA complex structure (Fig. 5A), CTD2 (the ASB binding site for lysine that would be transferred to the catalytic Ile2 domain) binds the acceptor helix of tRNA in the major groove center through the tunnel to react with the adenylated C34 side. CTD2 contains two antiparallel b-sheets with an inserted b- intermediate. 276 T. Suzuki, K. Miyauchi / FEBS Letters 584 (2010) 272–277

CTD2 A (ASB domain) B

Lys440

Lys404 Ile2 Glu412 tRNA HTH CTD1 Lys414 motif Arg394 (SCL domain) C4 Ile411 Lys407 Lys405

G69 Arg420 Arg389

C5 α-helical G68 Arg391 linker C Leu277 NTD A38 Arg142 binding pocket

C32 A38 Leu278

A37 Ser144 U36 Thr198 A35 Gln100 Arg156

Arg199 C34 Glu134 Arg165 His127 AT P Asp131

P-loop

Lysine

Fig. 5. Crystal structure of the TilS–tRNA complex. (A) Overall structure of the GkTilS–tRNA complex. The atomic coordinates (3A2K) were obtained from the Protein Data Bank. The three-dimensional structures were displayed using DS Visualizer (Accelrys). (B) Recognition of the acceptor helix by the HTH motif in CTD2. (C) Recognition of the anticodon loop by NTD. The sites for binding of ATP and lysine are colored in orange and red, respectively.

The entire process of tRNA recognition and lysidine formation able from the public database (http://www.ebi.ac.uk/genomes/ by type-I TilS is proposed to take place as follows [20]. The HTH bacteria.html), no homologs of tilS are found in 3 species of bacte- motif in CTD2 interacts with the acceptor helix of tRNAIle2, mainly ria, Mycoplasma mobile, Bifidobacterium adolescentis and Neorickett- by recognizing two consecutive C-G pairs (C4-G69 and C5-G68). sia sennetsu str. Miyayama. In these species, tDNAsIle with TAT This interaction disrupts the hydrophobic interaction between anticodon are present, suggesting AUA codons are deciphered by the HTH motif and CTD1. Following this recognition, the anticodon tRNAIleUAU instead of tRNAIleLAU. In addition, tDNAsIle bearing a loop of tRNAIle2 docks on the crater of the NTD, causing a confor- TAT anticodon are also encoded in the genomes of Rhodopirellula mational change of the anticodon loop, which ejects A38. The baltica SH-1 and Lactobacillus casei BL23.AstilS homologs are pres- hydrophobic pocket of CTD1 captures the ejected A38 as it grasps ent in these two species, AUA codons are redundantly deciphered to the edge of the anticodon loop. This conformational change in by tRNAIleLAU and tRNAIleUAU. Although a few bacteria do not em- the anticodon loop places C34 at the catalytic center. Then, the ploy TilS/Lysidine system to decode AUA codons, almost major spe- C-2 carbonyl group of C34 is adenylated by a pyrophosphatase-like cies of bacteria including pathogenic microbes decode AUA codons reaction to form the adenylated intermediate, which is subjected by tRNAIleLAU. to nucleophilic attack by the e-amino group of lysine to complete No homolog of TilS has been identified in mammalian genomes. the formation of lysidine. This was to be expected, because the AUA codon in the eukaryotic cytoplasm is deciphered by tRNAsIle bearing inosine or pseudouri- 7. TilS is a potential target for antibacterial drugs dine at their wobble positions. In mammalian mitochondrial decoding system, the AUA codon is read as Met by tRNA bearing 5 TilS is a highly conserved essential enzyme in eubacteria. In the f CAU anticodon [21]. Lysidine/TilS is a eubacterial-specific over 1000 species of bacteria whose genomic sequences are avail- system of AUA decoding. Therefore, TilS is an ideal target for a T. Suzuki, K. Miyauchi / FEBS Letters 584 (2010) 272–277 277 broad-spectrum antibacterial agent. In fact, a series of lysine ana- [11] Bork, P. and Koonin, E.V. (1994) A P-loop-like motif in a widespread ATP logs were tested in an effort to identify lead compounds that pyrophosphatase domain: implications for the evolution of sequence motifs and enzyme activity. Proteins 20, 347–355. potentially inhibit EcTilS [22]. The accumulating biochemical and [12] Ikeuchi, Y., Soma, A., Ote, T., Kato, J., Sekine, Y. and Suzuki, T. 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