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A major challenge in the field of RNA chemistry is the identification of selective and quantitative conversion reactions on RNA that can be used for tagging and any other RNA tool development. Here, we introduce metal‐free diazotransfer on native RNA containing an aliphatic primary amino group using the diazotizing reagent fluorosulfuryl azide (FSO2N3). The reaction provides the corresponding azide‐modified RNA in nearly quantitatively yields without affecting the nucleobase amino groups. The obtained azido‐RNA can then be further processed utilizing well‐established bioortho‐gonal reactions, such as azide‐alkyne cycloadditions (Click) or Staudinger ligations. We exemplify the robustness of this approach for the synthesis of peptidyl‐tRNA mimics and for the pull‐down of 3‐(3‐amino‐3‐carboxypropyl)uridine (acp3U)‐ and lysidine (k2C)‐containing tRNAs of an Escherichia coli tRNA pool isolated from cellular extracts. Our approach therefore adds a new dimension to the targeted chemical manipulation of diverse RNA species., Metal‐free diazotransfer on native RNA using the reagent FSO2N3 selectively provides azide‐modified RNA in quantitative yields. The stereochemically controlled reaction opens new avenues for the synthesis of RNA‐peptide conjugates and for the isolation of cellular RNA containing post‐transcriptional nucleotide modifications with an aliphatic primary amino group, such as acp3U.

Nucleotides of transfer RNAs (tRNAs) are highly modified, particularly at the anticodon. Bacterial tRNAs that read A-ending codons are especially notable. The U34 nucleotide canonically present in these tRNAs is modified by a wide range of complex chemical constituents. An additional two A-ending codons are not read by U34-containing tRNAs but are accommodated by either inosine or lysidine at the wobble position (I34 or L34). The structural basis for many N34 modifications in both tRNA aminoacylation and ribosome decoding has been elucidated, and evolutionary conservation of modifying enzymes is also becoming clearer. Here we present a brief review of the structure, function, and conservation of wobble modifications in tRNAs that translate A-ending codons. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1158-1166, 2019.

Lysidine (k2C) is one of the most modified pyrimidine RNA bases. It is a cytidine nucleoside, in which the 2‐oxo functionality of the heterocycle is replaced by the ϵ‐amino group of the amino acid lysine. As such, lysidine is an amino acid‐containing RNA nucleoside that combines directly genotype (C‐base) with phenotype (lysine amino acid). This makes the compound particularly important in the context of theories about the origin of life and here especially for theories that target the origin of translation. Here, we report the total synthesis of the U‐derivative of lysidine (k2U), which should have the same base pairing characteristics as k2C if it exists in the isoC‐like tautomeric form. To investigate this question, we developed a phosphoramidite building block for k2U, which allows its incorporation into RNA strands. Within RNA, k2U can base pair with the counter base U and isoG, confirming that k2U prefers an isoC‐like tautomeric structure that is also known to dominate for k2C. The successful synthesis of a k2U phosphoramidite and its use for RNA synthesis now paves the way for the preparation of a k2C phosphoramidite and RNA strands containing k2C.

Phages are the most abundant biological entity on Earth. There are many variants in phage virion sizes, morphology, and genome sizes. Large virion sized phages, with genome sizes greater than 200 kbp have been identified and termed as Jumbo phages. These phages exhibit certain characteristics that have not been reported in phages with smaller genomes. In this work, a jumbo phage named MIJ3 (vB_PaeM_MIJ3) that infects Pseudomonas aeruginosa PAO1 was isolated from an equine livery yard in Leicestershire, United Kingdom. The genome and biological characteristics of this phage have been investigated. MIJ3 is a Myovirus with multiple long tail fibers. Assessment of the host range of MIJ3 revealed that it has the ability to infect many clinical isolates of P. aeruginosa. Bioinformatics analysis of the phage genome indicated that MIJ3 is closely related to the Pseudomonas phage, PA5oct. MIJ3 possesses several unusual features that are either rarely present in other phages or have not yet been reported. In particular, MIJ3 encodes a FtsH-like protein, and a putative lysidine synthase, TilS. These two proteins have not been reported in phages. MIJ3 also possesses a split DNA polymerase B with a novel intein. Of particular interest, unlike other jumbo phages infecting Pseudomonas spp., MIJ3 lacks the genetic elements required for the formation of the phage nucleus, which was believed to be conserved across jumbo Pseudomonas phages.

Anhydrouridines react with aliphatic amines to give N-alkyl isocytosines, but reported procedures often demand very long reaction times and can be low yielding, with narrow scope. A modified procedure for such reactions has been developed, using microwave irradiation, significantly reducing reaction time and allowing facile access to a diverse range of novel nucleosides on the gram scale. The method has been used to prepare a precursor to a novel analogue of lysidine, a naturally occurring iminonucleoside found in (t)RNA.

Viruses rely on their host&rsquo, s translation machinery for the synthesis of their own proteins. Problems belie viral translation when the host has a codon usage bias (CUB) that is different from an infecting virus due to differences in the GC content between the host and virus genomes. Here, we examine the hypothesis that chloroviruses adapted to host CUB by acquisition and selection of tRNAs that at least partially favor their own CUB. The genomes of 41 chloroviruses comprising three clades, each infecting a different algal host, have been sequenced, assembled and annotated. All 41 viruses not only encode tRNAs, but their tRNA genes are located in clusters. While differences were observed between clades and even within clades, seven tRNA genes were common to all three clades of chloroviruses, including the tRNAArg gene, which was found in all 41 chloroviruses. By comparing the codon usage of one chlorovirus algal host, in which the genome has been sequenced and annotated (67% GC content), to that of two of its viruses (40% GC content), we found that the viruses were able to at least partially overcome the host&rsquo, s CUB by encoding tRNAs that recognize AU-rich codons. Evidence presented herein supports the hypothesis that a chlorovirus tRNA cluster was present in the most recent common ancestor (MRCA) prior to divergence into three clades. In addition, the MRCA encoded a putative isoleucine lysidine synthase (TilS) that remains in 39/41 chloroviruses examined herein, suggesting a strong evolutionary pressure to retain the gene. TilS alters the anticodon of tRNAMet that normally recognizes AUG to then recognize AUA, a codon for isoleucine. This is advantageous to the chloroviruses because the AUA codon is 12&ndash, 13 times more common in the chloroviruses than their host, further helping the chloroviruses to overcome CUB. Among large DNA viruses infecting eukaryotes, the presence of tRNA genes and tRNA clusters appear to be most common in the Phycodnaviridae and, to a lesser extent, in the Mimiviridae.

Hypermodified nucleosides lysidine (L) and N(6)-threonylcarbamoyladenosine (t(6)A) influence codon-anticodon interactions during the protein biosynthesis process. Lysidine prevents the misrecognition of the AUG codon as isoleucine and that of AUA as methionine. The structural significance of these modified bases has not been studied in detail at the atomic level. Hence, in the present study we performed multiple molecular dynamics (MD) simulations of anticodon stem loop (ASL) of tRNA(Ile) in the presence and absence of modified bases 'L' and 't(6)A' at the 34th and 37th positions respectively along with trinucleotide 'AUA' and 'AUG' codons. Hydrogen bonding interactions formed by the tautomeric form of lysidine may assist in reading the third base adenine of the 'AUA' codon, unlike the guanine of the 'AUG' codon. Such interactions might be useful to restrict codon specificity to recognize isoleucine tRNA instead of methionine tRNA. The t(6)A side chain interacts with the purine ring of the first codon nucleotide adenine, which might provide base stacking interactions and could be responsible for restricting extended codon-anticodon recognition. We found that ASL tRNA(Ile) in the absence of modifications at the 34th and 37th positions cannot establish proper hydrogen bonding interactions to recognize the isoleucine codon 'AUA' and subsequently disturbs the anticodon loop structure. The binding free energy calculations revealed that tRNA(Ile) ASL with modified nucleosides prefers the codon AUA over AUG. Thus, these findings might be useful to understand the role of modified bases L and t(6)A to recognize the AUA codon instead of AUG.

Modification of tRNA anticodons plays a critical role in ensuring accurate translation. N4-acetylcytidine (ac4C) is present at the anticodon first position (position 34) of bacterial elongator tRNAMet. Herein, we identified Bacillus subtilis ylbM (renamed tmcAL) as a novel gene responsible for ac4C34 formation. Unlike general acetyltransferases that use acetyl-CoA, TmcAL activates an acetate ion to form acetyladenylate and then catalyzes ac4C34 formation through a mechanism similar to tRNA aminoacylation. The crystal structure of TmcAL with an ATP analog reveals the molecular basis of ac4C34 formation. The ΔtmcAL strain displayed a cold-sensitive phenotype and a strong genetic interaction with tilS that encodes the enzyme responsible for synthesizing lysidine (L) at position 34 of tRNAIle to facilitate AUA decoding. Mistranslation of the AUA codon as Met in the ΔtmcAL strain upon tilS repression suggests that ac4C34 modification of tRNAMet and L34 modification of tRNAIle act cooperatively to prevent misdecoding of the AUA codon.

Translation of the isoleucine codon AUA in most prokaryotes requires a modified C (lysidine or agmatidine) at the wobble position of tRNA2(Ile) to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the essential gene encoding tRNA(Ile)-lysidine synthetase was deleted for the first time. In such a strain, C34 at the wobble position of tRNA2(Ile) is expected to remain unmodified and cells depend on a mutant suppressor tRNA derived from tRNA1(Ile), in which G34 has been changed to U34. An important question, therefore, is how U34 base pairs with A without also base pairing with G. Here, we show (i) that unlike U34 at the wobble position of all B. subtilis tRNAs of known sequence, U34 in the mutant tRNA is not modified, and (ii) that the mutant tRNA binds strongly to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data explain why the suppressor strain displays only a low level of misreading AUG codons in vivo and, as shown here, grows at a rate comparable to that of the wild-type strain.

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel 'readers' of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and their use in biotechnology.

In thermophilic bacteria, specific 2-thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2-thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur-carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2-thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 A resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N- and C-terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNAIle2 lysidine synthetase), than to the other type of tRNA 2-thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP-binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA-binding site. Proteins 2013; 81:1232–1244. © 2013 Wiley Periodicals, Inc.

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.

In eubacteria, the post-transcriptional modification of the wobble cytidine of the CAU anticodon in a precursor tRNA(Ile2) to a lysidine residue (2-lysyl-cytidine, abbreviated as L) allows the amino acid specificity to change from methionine to isoleucine and the codon decoding specificity to shift from AUG to AUA. The tilS gene encoding the enzyme that catalyses this modification is widely distributed. However, some microbial species lack a tilS gene, indicating that an alternative strategy exists to accurately translate the AUA codon into Ile. To determine whether a TilS-dependent bacterium, such as Bacillus subtilis, can overcome the absence of lysidine in its tRNA(Ile2) (CAU), we analysed the suppressor mutants of a tilS-thermosensitive allele. These tilS-suppressor mutants carry a substitution of the wobble guanosine into thymidine in one of the tRNA(Ile1) genes (the original GAT anticodon is changed to a TAT). In absence of TilS activity, the AUA codons are translated into isoleucine by the suppressor tRNA(Ile1), although a low level of AUA codons is also mistranslated into methionine. Results are in agreement with rare cases of eubacteria (and archaea), which naturally lack the tilS gene (or tiaS in archaea) but contain a tRNA(Ile2) gene containing a TAT instead of a CAT anticodon.

International audience; The preparation of first lysidinyl-triphosphine ligand (named Triphosline) is described in three steps which are first a Michael type addition of imidazolidine (or lysidine) to diethylvinylphosphonate, second a phosphonate reduction with LiAlH4 and third an anti-Markovnikov radical addition of the primary phosphine to diphenylvinylphosphine. The Triphosline behaves as a tridentate P-coordinating ligand in palladium(II) complexes. The dangling lysidine function is then cleanly and totally alkylated by methyl iodide to lead to a new kind of lysidinium-triphosphine complexes. Subsequent anion exchange with TlPF6 affords the first example of a chloride free lysidinium-triphosphine palladium complex which has been fully characterized by spectroscopic and analytical methods.

Transfer RNAs are transcribed as precursor molecules which are then shortened, have a short sequence added, and can undergo recoding of certain nucleotides to generate a different amino acid specificity. In this work, Nakanishi et al. show how the bacterial enzyme TilS uniquely recognizes a precursor tRNA that has a specificity for methionine and then introduces a lysidine-for-cytidine modification in the anticodon that results in the mature tRNA coding for isoleucine. They find that by acting upon the precursor tRNA, TilS thereby prevents charging of the tRNA by the methionyl-tRNA synthetase, which only acts on a mature tRNA. This mechanism contributes to the vital goal of maintaining the fidelity of isoleucine codon translation in bacteria. tRNAs are transcribed as precursor molecules that are then shortened, have a short sequence added, and may then undergo modifications of certain nucleotides to generate a different amino acid specificity. Here, tRNAIle2 lysidine synthetase (TilS) — a bacterial enzyme that carries out a nucleotide modification — is shown to specifically recognize and modify tRNAIle2 in its precursor form, thereby avoiding potential translation errors. Maturation of precursor transfer RNA (pre-tRNA) includes excision of the 5′ leader and 3′ trailer sequences, removal of introns and addition of the CCA terminus1,2,3. Nucleotide modifications are incorporated at different stages of tRNA processing, after the RNA molecule adopts the proper conformation. In bacteria, tRNAIle2 lysidine synthetase (TilS) modifies cytidine into lysidine (L; 2-lysyl-cytidine) at the first anticodon of tRNAIle2 (refs 4–9). This modification switches tRNAIle2 from a methionine-specific to an isoleucine-specific tRNA9. However, the aminoacylation of tRNAIle2 by methionyl-tRNA synthetase (MetRS), before the modification by TilS, might lead to the misincorporation of methionine in response to isoleucine codons. The mechanism used by bacteria to avoid this pitfall is unknown. Here we show that the TilS enzyme specifically recognizes and modifies tRNAIle2 in its precursor form, thereby avoiding translation errors. We identified the lysidine modification in pre-tRNAIle2 isolated from RNase-E-deficient Escherichia coli and did not detect mature tRNAIle2 lacking this modification. Our kinetic analyses revealed that TilS can modify both types of RNA molecule with comparable efficiencies. X-ray crystallography and mutational analyses revealed that TilS specifically recognizes the entire L-shape structure in pre-tRNAIle2 through extensive interactions coupled with sequential domain movements. Our results demonstrate how TilS prevents the recognition of tRNAIle2 by MetRS and achieves high specificity for its substrate. These two key points form the basis for maintaining the fidelity of isoleucine codon translation in bacteria. Our findings also provide a rationale for the necessity of incorporating specific modifications at the precursor level during tRNA biogenesis.

Bacteria decode the isoleucine codon AUA using a tRNA species that is posttranscriptionally modified at the wobble position of the anticodon with a lysine-containing cytidine derivative called lysidine. The lysidine modification of tRNAIle2 is an essential identity determinant for proper aminoacylation by isoleucyl tRNA synthetase (IleRS) and codon recognition on the ribosome. The ATP- and lysine-dependent formation of lysidine is catalyzed by tRNAIle-lysidine synthetase. Using the purified recombinant enzyme from Escherichia coli and an in vitro transcribed tRNA substrate, we have confirmed that lysidine modification is both necessary and sufficient to convert tRNAIle2 into a substrate for IleRS. A series of lysine analogs were tested as potential inhibitors during the mechanistic characterization of tRNAIle-lysidine synthetase. Gel electrophoresis revealed that many of these analogs, including some simple alkyl amines, were alternative substrates. Incorporation of these amines into alternative tRNA products was confirmed by mass spectrometry. The availability of tRNAIle2 with differential modifications enabled an exploration of the structural requirements of the anticodon for aminoacylation by methionyl tRNA synthetase and IleRS. All of the modifications were effective at creating negative determinants for methionyl tRNA synthetase and positive determinants for IleRS, although the tolerance of IleRS differed between the enzymes from E. coli and Bacillus subtilis.

Conformational preferences of hypermodified nucleoside, 4-amino-2-(N(6)-lysino)-1-(beta-D-ribofuranosyl) pyrimidinium (Lysidine or 2-lysyl cytidine), usually designated as k(2)C, have been investigated theoretically by the quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. The zwitterionic, non-zwitterionic, neutral, and tautomeric forms have been studied. Automated geometry optimization using molecular mechanics force field (MMFF), semi-empirical quantum chemical PM3, and ab initio molecular orbital Hartree-Fock SCF quantum mechanical calculations have also been made to compare the salient features. The predicted most stable conformations of zwitterionic, non-zwitterionic, neutral, and tautomeric form are such that in each of these molecules the orientation of lysidine moiety (R) is trans to the N(1) of cytidine. The preferred base orientation is anti (chi = 3 degrees ) and the lysine substituent folds back toward the ribose ring. This results in hydrogen bonding between the carboxyl oxygen O(12a) of lysine moiety and the 2'-hydroxyl group of ribose sugar. In all these four forms of lysidine O(12a)...H-C(9) and O(12b)...H-N(11) interactions provide stability to respective stable conformers. Watson-Crick base pairing of lysidine with A is feasible only with the tautomeric form of usual anti oriented lysidine. This can help in recognition of AUA codon besides in avoiding misrecognition of AUG.

Catalytic ammonolysis of 2-methylimidazoline (lysidine) in the presence of a number of ternary molybdenum-antimony-titanium oxide systems was studied.

Lysidine (2-lysyl cytidine) is a lysine-containing cytidine derivative commonly found at the wobble position of bacterial AUA codon-specific tRNA(Ile). This modification determines both codon and amino acid specificities of tRNA(Ile). We previously identified tRNA(Ile)-lysidine synthetase (tilS) that synthesizes lysidine, for which it utilizes ATP and lysine as substrates. Here, we show that lysidine synthesis consists of two consecutive reactions that involve an adenylated tRNA intermediate. A mutation study revealed that Escherichia coli TilS discriminates tRNA(Ile) from the structurally similar tRNA(Met) having the same anticodon loop by recognizing the anticodon loop, the anticodon stem, and the acceptor stem. TilS was shown to bind to the anticodon region and 3' side of the acceptor stem, which cover the recognition sites. These findings reveal a dedicated mechanism embedded in tRNA(Ile) that controls its recognition and discrimination by TilS, and indicate the significance of this enzyme in the proper deciphering of genetic information.

Lysidine, a lysine-combined modified cytidine, is exclusively located at the anticodon wobble position (position 34) of eubacterial tRNA Ile 2 and not only converts the codon specificity from AUG to AUA, but also converts the aminoacylation specificity from recognition by methionyl-tRNA synthetase to that by isoleucyl-tRNA synthetase (IleRS). Here, we report the crystal structure of lysidine synthetase (TilS) from Aquifex aeolicus at 2.42-Å resolution. TilS forms a homodimer, and each subunit consists of the N-terminal dinucleotide-binding fold domain (NTD), with a characteristic central hole, and the C-terminal globular domain (CTD) connected by a long α-helical linker. The NTD shares striking structural similarity with the ATP-pyrophosphatase domain of GMP synthetase, which reminds us of the two-step reaction by TilS: adenylation of C34 and lysine attack on the C2 carbon. Conserved amino acid residues are clustered around the NTD central hole. Kinetic analyses of the conserved residues' mutants indicated that C34 of tRNA Ile 2 is adenylated by an ATP lying across the NTD central hole and that a lysine, which is activated at a loop appended to the NTD, nucleophilically attacks the C2 carbon from the rear. Escherichia coli TilS (called MesJ) has an additional CTD, which may recognize the tRNA Ile 2 acceptor stem. In contrast, a mutational study revealed that A. aeolicus TilS does not recognize the tRNA acceptor stem but recognizes the C29·G41 base pair in the anticodon stem. Thus, the two TilS enzymes discriminate tRNA Ile 2 from tRNA Met by strategies similar to that used by IleRS, but in distinct manners.

In most organisms, the AUA triplet codes for isoleucine (Ile), whereas in a few organelles it codes for methionine (Met). In bacteria, this A-ending triplet is decoded by an unusual tRNA harboring a Met anticodon CAU, where cytidine at the wobble position 34 (C34) is posttranscriptionally modified to a 2-lysyl cytidine (lysidine), abbreviated as (k2C). Now, the bacterial gene tilS, which encodes the enzyme catalyzing the lysylation of C34 in the precursor tRNAIle(CAU), thereby leading to the formation of tRNAIle(k2CAU), has been identified. The formation of lysidine by this essential enzyme allows recognition of tRNAIle(k2CAU) by Ile-tRNA synthetase and switches the base pairing of the tRNA from AUG (Met) to AUA (Ile). This base change is reminiscent of C-to-U type of RNA editing of some mitochondrial tRNAs.

The AUA codon-specific isoleucine tRNA (tRNA Ile ) in eubacteria has the posttranscriptionally modified nucleoside lysidine (L) at the wobble position of the anticodon (position 34). This modification is a lysine-containing cytidine derivative that converts both the codon specificity of tRNA Ile from AUG to AUA and its amino acid specificity from methionine to isoleucine. We identified an essential gene ( tilS ; tRNA Ile -lysidine synthetase) that is responsible for lysidine formation in both Bacillus subtilis and Escherichia coli . The recombinant enzyme complexed specifically with tRNA Ile and synthesized L by utilizing ATP and lysine as substrates. The lysidine synthesis of this enzyme was shown to directly convert the amino acid specificity of tRNA Ile from methionine to isoleucine in vitro. Partial inactivation of tilS in vivo resulted in an AUA codon-dependent translational defect, which supports the notion that TilS is an RNA-modifying enzyme that plays a critical role in the accurate decoding of genetic information.

From 50 genomes of the three domains of life (7 eukarya, 13 archaea, and 30 bacteria), we extracted, analyzed, and compared over 4,000 sequences corresponding to cytoplasmic, nonorganellar tRNAs. For each genome, the complete set of tRNAs required to read the 61 sense codons was identified, which permitted revelation of three major anticodon-sparing strategies. Other features and sequence peculiarities analyzed are the following: (1) fit to the standard cloverleaf structure, (2) characteristic consensus sequences for elongator and initiator tDNAs, (3) frequencies of bases at each sequence position, (4) type and frequencies of conserved 2D and 3D base pairs, (5) anticodon/tDNA usages and anticodon-sparing strategies, (6) identification of the tRNA-Ile with anticodon CAU reading AUA, (7) size of variable arm, (8) occurrence and location of introns, (9) occurrence of 3'-CCA and 5'-extra G encoded at the tDNA level, and (10) distribution of the tRNA genes in genomes and their mode of transcription. Among all tRNA isoacceptors, we found that initiator tDNA-iMet is the most conserved across the three domains, yet domain-specific signatures exist. Also, according to which tRNA feature is considered (5'-extra G encoded in tDNAs-His, AUA codon read by tRNA-Ile with anticodon CAU, presence of intron, absence of "two-out-of-three" reading mode and short V-arm in tDNA-Tyr) Archaea sequester either with Bacteria or Eukarya. No common features between Eukarya and Bacteria not shared with Archaea could be unveiled. Thus, from the tRNomic point of view, Archaea appears as an "intermediate domain" between Eukarya and Bacteria.

Bacteria and archaea have 2-lysylcytidine (L or lysidine) and 2-agmatinylcytidine (agm2C or agmatidine), respectively, at the first (wobble) position of the anticodon of the AUA codon-specific tRNAIle. These lysine- or agmatine-conjugated cytidine derivatives are crucial for the precise decoding of the genetic code. L is synthesized by tRNAIle-lysidine synthetase (TilS), which uses l-lysine and ATP as substrates. Agm2C formation is catalyzed by tRNAIle-agm2C synthetase (TiaS), which uses agmatine and ATP for the reaction. Despite the fact that TilS and TiaS synthesize structurally similar cytidine derivatives, these enzymes belong to non-related protein families. Therefore, these enzymes modify the wobble cytidine by distinct catalytic mechanisms, in which TilS activates the C2 carbon of the wobble cytidine by adenylation, while TiaS activates it by phosphorylation. In contrast, TilS and TiaS share similar tRNA recognition mechanisms, in which the enzymes recognize the tRNA acceptor stem to discriminate tRNAIle and tRNAMet.

A novel, ultrahigh-throughput, fluorescence anisotropy-based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNA(Ile) lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to eliminate false positives in the initial screen resulting from detection artifacts as well as compounds competitive with the fluorescent label instead of ATP, and to measure inhibitor potencies against E. coli and Pseudomonas aeruginosa TilS isozymes. The tRNA(Ile) substrate for P. aeruginosa TilS was identified for the first time to enable these measurements. ATP-competitive binding of inhibitors was confirmed by one-dimensional ligand-observe nuclear magnetic resonance. A preliminary structure-activity relationship is shown for two inhibitor series.

Recognition strategies for tRNA aminoacylation are ancient and highly conserved, having been selected very early in the evolution of the genetic code. In most cases, the trinucleotide anticodons of tRNA are important identity determinants for aminoacylation by cognate aminoacyl-tRNA synthetases. However, a degree of ambiguity exists in the recognition of certain tRNA(Ile) isoacceptors that are initially transcribed with the methionine-specifying CAU anticodon. In most organisms, the C34 wobble position in these tRNA(Ile) precursors is rapidly modified to lysidine to prevent recognition by methionyl-tRNA synthetase (MRS) and production of a chimeric Met-tRNA(Ile) that would compromise translational fidelity. In certain bacteria, however, lysidine modification is not required for MRS rejection, indicating that this recognition strategy is not universally conserved and may be relatively recent. To explore the actual distribution of lysidine-dependent tRNA(Ile) rejection by MRS, we have investigated the ability of bacterial MRSs from different clades to differentiate cognate tRNACAU(Met) from near-cognate tRNACAU(Ile). Discrimination abilities vary greatly and appear unrelated to phylogenetic or structural features of the enzymes or sequence determinants of the tRNA. Our data indicate that tRNA(Ile) identity elements were established late and independently in different bacterial groups. We propose that the observed variation in MRS discrimination ability reflects differences in the evolution of genetic code machineries of emerging bacterial clades.

Deciphering the genetic code is a fundamental process in all living organisms. In many bacteria, AUA codons are deciphered by tRNA(Ile2) bearing lysidine (L) at the wobble position. L is a modified cytidine introduced post-transcriptionally by tRNA(Ile)-lysidine synthetase (TilS). Some bacteria, including Mycoplasma mobile, do not carry the tilS gene, indicating that they have established a different system to decode AUA codons. In this study, tRNA(Ile2) has been isolated from M. mobile and was found to contain a UAU anticodon without any modification. Mycoplasma mobile isoleucyl-tRNA synthetase (IleRS) recognized the UAU anticodon, whereas Escherichia coli IleRS did not efficiently aminoacylate tRNA(Ile2)(UAU). In M. mobile IleRS, a single Arg residue at position 865 was critical for specificity for the UAU anticodon and, when the corresponding site (W905) in E. coli IleRS was substituted with Arg, the W905R mutant efficiently aminoacylated tRNA with UAU anticodon. Mycoplasma mobile tRNA(Ile2) cannot distinguish between AUA and AUG codon on E. coli ribosome. However, on M. mobile ribosome, M. mobile tRNA(Ile2)(UAU) specifically recognized AUA codon, and not AUG codon, suggesting M. mobile ribosome has a property that prevents misreading of AUG codon. These findings provide an insight into the evolutionary reorganization of the AUA decoding system.

Escherichia coli tRNAIle 1 prepared in vitro by T7 RNA polymerase transcription was found to be charged with isoleucine by E. coli isoleucyl-tRNA synthetase (IleRS). Replacement of G in the first position of the anticodon with U, C, or A resulted in complete loss of isoleucine-accepting activity. This indicates that IleRS strictly recognizes the structural feature of guanosine in this position of tRNAIle 1 which is common to that of lysidine in E. coli tRNAIle 2.

Annotation of the complete genome of the extreme halophilic archaeon Haloarcula marismortui does not include a tRNA for translation of AUA, the rare codon for isoleucine. This is a situation typical for most archaeal genomes sequenced to date. Based on computational analysis, it has been proposed recently that a single intron-containing tRNA gene produces two very similar but functionally different tRNAs by means of alternative splicing; a UGG-decoding tRNATrpCCA and an AUA-decoding tRNAIleUAU. Through analysis of tRNAs from H. marismortui, we have confirmed the presence of tRNATrpCCA, but found no evidence for the presence of tRNAIleUAU. Instead, we have shown that a tRNA, currently annotated as elongator methionine tRNA and containing CAU as the anticodon, is aminoacylated with isoleucine in vivo and that this tRNA represents the missing isoleucine tRNA. Interestingly, this tRNA carries a base modification of C34 in the anticodon different from the well-known lysidine found in eubacteria, which switches the amino acid identity of the tRNA from methionine to isoleucine and its decoding specificity from AUG to AUA. The methods described in this work for the identification of individual tRNAs present in H. marismortui provide the tools necessary for experimentally confirming the presence of any tRNA in a cell and, thereby, to test computational predictions of tRNA genes.

Imidazoline, a highly basic amine, was employed as a functional group in weak-base resins that were prepared for the extraction of gold cyanide from alkaline cyanide leach liquors. The chemical stability of the resins and their model compound analogues under these conditions was investigated. When the imidazoline group was attached to an acrylonitrile and a polystyrene matrix, it hydrolysed slowly to form an amide group. The attachment of lysidine (2-methylimidazoline) to a polystyrene matrix yielded a resin in which the hydrolysis product underwent a second hydrolysis to form an alkylamine group on the resin. The relative rates of hydrolysis of the resins were confirmed by an investigation using NMR spectroscopy of the hydrolysis reactions of the model compound analogues of the resins, i.e., lysidine (2-methylimidazoline), benzylimidazoline, and benzyllysidine (2-methylbenzylimidazoline). The hydrolysis products were the same as those for the resins; the rates of hydrolysis for the different structures increased in the order lysidine, benzylimidazoline, benzyllysidine. The model compounds are more rapidly hydrolysed than the resins-50% of the functional groups of the acrylonitrile-based imidazoline resin hydrolysed during four years of contact with an alkaline solution, whereas the model compound of this resin hydrolysed completely over a period of 320 hours.

The crystal structure of lysidine · HAu(CN)2 was determined so that the influence of steric hindrance at the active site of weak-base imidazoline resins could be established. These resins, which have alkyl groups attached to the basic nitrogen, have lower basicities, and are also eluted more slowly, than imidazoline resins that have no substituents at the active site. The crystal structure of lysidine · HAu(CN)2 (a model compound for one of the unsubstituted resins) consists of two interpenetrating hydrogen-bonded networks that are perpendicular to each other. The structure suggests that the presence of an alkyl substituent on the basic nitrogen of an imidazoline weak-base resin could hinder the approach of a hydrogen atom and the gold cyanide anion to the basic nitrogen in the functional group, thus lowering the basicity of the amine. However, the crystal structure does not explain the steric effects that have been postulated as causing the rate of elution of disubstituted imidazoline resins to be slower than that of monosubstituted imidazoline resins.

Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs and aminoacyl-tRNA synthetases. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA(Pro) with cysteine, but does not aminoacylate archaeal tRNA(Cys) with cysteine; (iv) identification and characterization of the AUA-decoding minor tRNA(Ile) in archaea; and (v) evidence that the archaeal minor tRNA(Ile) contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.

The structural significance of the hypermodified nucleosides lysidine (k2C) and N6-(N-threonylcarbonyl) adenosine (t6A) present in the anticodon loop of E. coli tRNAIle has not been studied in detail theoretically at the atomic level. Hence, in the present paper, we investigated the conformational preferences of modified nucleosides k2C and t6A in the anticodon loop of tRNAIle using various quantum chemical methods. Multiple molecular dynamics simulation studies of the anticodon stem loop (ASL) of tRNAIle have also been made to see the solvation effect. The lysine moiety of lysidine orients back and forms a hydrogen bond with the 2′-hydroxyl group of the 34th ribose sugar whereas the t6A(37) side chain prefers a ‘distal’ conformation. This kind of conformation would be useful for recognition of the codons AUA instead of AUG. The t6A side chain prohibits canonical base pairing and maintains an anticodon open loop structure. The preferred conformations of k2C(34) and t6A(37) are stabilized by various intra as well as inter residual interactions within the anticodon loop which could be useful for maintaining a proper anticodon loop structure for smooth and in phase codon–anticodon interactions during the biosynthesis of proteins.

Earlier work by two independent groups has established the fact that anticodons GAU and LAU of Escherichia coli tRNAIle isoacceptors play a critical role in the tRNA identity. Yeast possesses two isoleucine transfer RNAs, a major one with anticodon IAU and a minor one with anticodon PsiAPsi which are derived from the post-transcriptional modification of AAU and UAU gene sequences, respectively. We present direct evidence which reveals that inosine is a positive determinant for yeast isoleucyl-tRNA synthetase. We also show that yeast tRNAMet with guanosine at the wobble position becomes aminoacylated with isoleucine while methionine acceptance is lost. As inosine and guanosine share the 6-keto and the N-1 hydrogen groups, this suggests that these hydrogen donor and acceptor groups are determinants for isoleucine specificity. The role of the minor tRNAIle anticodon pseudouridines in tRNA isoleucylation could not be tested directly but was deduced from a 40-fold decrease in the activity of the unmodified transcript. The presence of the NHCO structure in guanosine, inosine, pseudouridine, and lysidine suggests a unifying model of wobble base recognition by the yeast and E. coli isoleucyl-tRNA synthetase. In contrast to lysidine which switches the identity of the tRNA from methionine to isoleucine [Muramatsu, T., Nishikawa, K., Nemoto, F., Kuchino, Y., Nishimura, S., Miyazawa, T., & Yokoyama, S. (1988) Nature 336, 179-181], pseudouridine-34 does not modify the specificity of the yeast minor tRNAIle since U-34 is a strong negative determinant for yeast MetRS. Therefore, the major role of Psi-34 (in combination with Psi-36 or not) is likely in isoleucine AUA codon specificity and translational fidelity.

Infrared and Raman spectroscopy is useful for studying helical conformations of polypeptides, which are determined by molecular structure parameters. Nuclear magnetic resonance spectroscopy, as well as X-ray analysis, is now established to be important for conformation studies of proteins and nucleic acids in solution. This article is mainly concerned with the conformational aspect and function regulation in protein biosynthesis. The strict recognition of transfer ribonucleic acid (tRNA) by aminoacyl-tRNA synthetase (ARS) is achieved by multi-step mutual adaptation. The conformations of ARS-bound amino acids have been elucidated by transferred nuclear Overhauser effect analysis. Aminoacyl-tRNA takes the 3′-isomeric form in the polypeptide chain elongation cycle. The regulation of codon recognition by post-transcriptional modification is achieved by conversion of the conformational characteristic of the anticodon of tRNA. The cytidine → lysidine modification of the anticodon of minor isoleucine tR...

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium ("lysidine"), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

It is suggested that while the standard base pairs may be used rather strictly in the first two positions of the triplet, there may be some wobble in the pairing of the third base. This hypothesis is explored systematically, and it is shown that such a wobble could explain the general nature of the degeneracy of the genetic code.

An Escherichia coli isoleucine transfer RNA specific for the codon AUA (tRNA2Ile or tRNAminorIle (ref. 1) has a novel modified nucleo-side, lysidine (L; ref. 2) (Fig. la) in the first position of the anticodon (position 34), which is essential for the specific recognition of the codon AUA (ref. 1). We isolated the gene for tRNA2Ile (ileX) and found that the anticodon is CAT, which is characteristic of the methionine tRNA gene. Replacement of L(34) of tRNA2Ile molecule enzymatically with unmodified C(34) resulted in a marked reduction of the isoleucine-accepting activity and, surprisingly, in the appearance of methionine-accepting activity. Thus, both the codon and amino-acid specificity of this tRNA are converted by a single post-transcriptional modification of the first position of the anticodon during tRNA maturation.

The reassignment of codon AUA from isoleucine to methionine during mitochondrial evolution may be explained by the codon reassignment (capture) hypothesis without assuming direct replacement of isoleucine by methionine in mitochondrial proteins. According to this hypothesis, codon AUA would have disappeared from the reading frames of messenger RNA. AUA codons would have mutated mainly to AUU isoleucine codons because of constraints resulting from elimination of tRNA Ile with anticodon *CAU (in which *C is lysidine). Later, tRNA Met (CAU) would have undergone structural changes enabling it to pair with both AUG and AUA. AUA codons, formed by mutations of other codons, including AUG, would have reappeared and would have been translated as methionine.

The nitration of lysidine with acetic anhydride, nitric acid, and a chloride catalyst does not yield the expected nitrolysidine. The product, instead, is 1,3-dinitroimidazolidone-2 when little or no chloride is used. A new compound is produced when a full equivalent of hydrogen chloride is included. This new compound is thought to be 1,3-dinitro-2-chloromethyl-2-acetoxylimidazolidine. Its decomposition products are ethylenedinitramine and chloroacetic acid. It is believed to be cyclic because the linear isomer, N-acetyl-N′-chloroacetylethylenedinitramine has been prepared and found not to be identical. The formation of dinitrochloromethylacetoxylimidazolidine in the nitration mixture suggests that it is formed by addition of chlorine acetate. This compound has been prepared, characterized, and found to add to allylbenzene. Since it will convert dicyclohexylamine to the chloramine it is postulated as a temporal ingredient of the catalyzed nitration mixture.

Transfer RNAs (tRNAs) are central players in translation, functioning as adapter molecules between the informational level of nucleic acids and the functional level of proteins. They show a highly conserved secondary and tertiary structure and the highest density of post-transcriptional modifications among all RNAs. These modifications concentrate in two hotspots—the anticodon loop and the tRNA core region, where the D- and T-loop interact with each other, stabilizing the overall structure of the molecule. These modifications can cause large rearrangements as well as local fine-tuning in the 3D structure of a tRNA. The highly conserved tRNA shape is crucial for the interaction with a variety of proteins and other RNA molecules, but also needs a certain flexibility for a correct interplay. In this context, it was shown that tRNA modifications are important for temperature adaptation in thermophilic as well as psychrophilic organisms, as they modulate rigidity and flexibility of the transcripts, respectively. Here, we give an overview on the impact of modifications on tRNA structure and their importance in thermal adaptation.

In most eubacteria, the minor AUA isoleucine codon is decoded by tRNAIle2, which has a lysidine (L) in the anticodon loop. The lysidine is introduced by tRNAIle-lysidine synthetase (TilS) through post-transcriptional modification of cytidine to yield an LAU anticodon. Some bacteria, Lactobacillus plantarum for example, possess two tRNAIle2(UAU) genes in addition to, two tRNAIle2(CAU) genes and the tilS gene. tRNA expression from all these genes would generate redundancy in a tRNA that decodes a rare AUA codon. In this study, we investigated the tRNA expression from these genes in L. plantarum and characterized the corresponding tRNAs. The tRNAIle2(CAU) gene products are modified by TilS to produce tRNAIle2(LAU), while tRNAIle2(UAU) lacks modification especially in the anticodon sequence. We found that tRNAIle2(LAU) is charged with isoleucine but tRNAIle2(UAU) is not. Our results suggest that the tRNAIle2 redundancy may be related to different roles of these tRNAs in the cell.