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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 tRNAIle 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 tRNAIle 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. [ABSTRACT FROM PUBLISHER]

Abstract: In the bacterial decoding system, the AUA codon is deciphered as isoleucine by tRNAIle bearing lysidine (L, 2-lysyl-cytidine) at the wobble position. Lysidine is an essential modification that determines both the codon and amino acid specificities of tRNAIle. We identified an enzyme named tRNAIle lysidine synthetase (TilS) that catalyzes lysidine formation by using lysine and ATP as substrates. Biochemical studies revealed a molecular mechanism of lysidine formation that consists 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 recognition by TilS, and a molecular basis of lysidine formation at atomic resolution. [Copyright &y& Elsevier]

Conformational preferences of hypermodified nucleoside, 4-amino-2-(N6-lysino)-1-(β-D-ribofuranosyl) pyrimidinium (Lysidine or 2-lysyl cytidine), usually designated as k2C, 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 (χ = 3°) 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. [ABSTRACT FROM AUTHOR]

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 spectros-copy revealed that the first letter of the anticodon is modified to lysidine [4-amino-2-(N6-lysino)-1-β-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. [ABSTRACT FROM AUTHOR]