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Discovery and Characterization of Trnaile Lysidine Synthetase (Tils)

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Discovery and Characterization of Trnaile Lysidine Synthetase (Tils) View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 584 (2010) 272–277 journal homepage: www.FEBSLetters.org 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 deci- phered how Escherichia coli TilS specifically discriminates between tRNAIle and the structurally sim- ilar 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].
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