anticodon and lysidine

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.

Topics: Amino Acyl-tRNA Synthetases; Anticodon; Bacillus; Bacteria; Base Pairing; Codon; Escherichia coli; Genetic Code; Inosine; Lysine; Models, Genetic; Mycobacterium; Protein Biosynthesis; Pyrimidine Nucleosides; Ribosomes; RNA Processing, Post-Transcriptional; RNA, Transfer; Thermus thermophilus

Deciphering AUA codons is a difficult task for organisms, because AUA and AUG specify isoleucine (Ile) and methionine (Met), separately. Each of the other purine-ending sense co-don sets (NNR) specifies a single amino acid in the universal genetic code. In bacteria and archaea, the cytidine derivatives, 2-lysylcytidine (L or lysidine) and 2-agmatinylcytidine (agm(2)C or agmatidine), respectively, are found at the first letter of the anticodon of tRNA(Ile) responsible for AUA codons. These modifications prevent base pairing with G of the third letter of AUG codon, and enable tRNA(Ile) to decipher AUA codon specifically. In addition, these modifications confer a charging ability of tRNA(Ile) with Ile. Despite their similar chemical structures, L and agm(2)C are synthesized by distinctive mechanisms and catalyzed by different classes of enzymes, implying that the analogous decoding systems for AUA codons were established by convergent evolution after the phylogenic split between bacteria and archaea-eukaryotes lineages following divergence from the last universal common ancestor (LUCA).

Topics: Anticodon; Archaea; Bacteria; Biological Evolution; Codon; Cytidine; Genetic Code; Isoleucine; Lysine; Methionine; Models, Molecular; Phylogeny; Protein Biosynthesis; Pyrimidine Nucleosides; Ribosomes; RNA, Transfer

Topics: Amino Acyl-tRNA Synthetases; Anticodon; Base Pairing; Cytidine; Escherichia coli Proteins; Isoleucine-tRNA Ligase; Lysine; Methionine-tRNA Ligase; Models, Chemical; Molecular Structure; Pyrimidine Nucleosides; RNA Editing; RNA, Transfer, Ile

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.

Topics: Adenosine; Anticodon; Base Sequence; Codon; Computer Simulation; Hydrogen Bonding; Lysine; Methionine; Molecular Dynamics Simulation; Molecular Sequence Data; Nucleosides; Pyrimidine Nucleosides; Ribosomes; Static Electricity

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.

Topics: Amino Acid Substitution; Amino Acyl-tRNA Synthetases; Anticodon; Bacillus subtilis; Hot Temperature; Lysine; Protein Biosynthesis; Pyrimidine Nucleosides; RNA, Transfer; Suppression, Genetic

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.

Topics: Anticodon; Base Pairing; Chemistry Techniques, Analytical; Hydrogen Bonding; Lysine; Molecular Structure; Nucleic Acid Conformation; Pyrimidine Nucleosides; RNA, Transfer, Ile

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-A 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 alpha-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.

Topics: Amino Acyl-tRNA Synthetases; Anticodon; Bacteria; Bacterial Proteins; Cloning, Molecular; Crystallography; Escherichia coli Proteins; Genetic Vectors; Kinetics; Lysine; Models, Molecular; Mutation; Protein Folding; Protein Structure, Tertiary; Pyrimidine Nucleosides; Pyrophosphatases; RNA, Transfer, Ile

Bacillus subtilis, which belongs to Gram-positive eubacteria, has been predicted to have a minor isoleucine tRNA transcribed from the gene possessing the CAT anticodon, which corresponds to methionine. We isolated this tRNA and determined its sequence including modified nucleotides. Modified nucleotide analyses using TLC, UV, and FAB mass spectroscopy revealed that the first letter of the anticodon is modified to lysidine [4-amino-2-(N6-lysino)-1-beta-d-ribofuranosyl pyrimidine]. As a result, this tRNA agrees with the minor one predicted from the DNA sequence and is thought to decode the isoleucine codon AUA.

Topics: Acylation; Anticodon; Bacillus subtilis; Base Sequence; DNA, Bacterial; Genes, Bacterial; Isoleucine; Lysine; Methionine; Molecular Sequence Data; Nucleic Acid Conformation; Pyrimidine Nucleosides; RNA, Transfer, Ile; Sequence Analysis, RNA; Transcription, Genetic