Modification of the cytidine in the first anticodon position of the AUA decoding tRNA Ile (tRNA Ile 2 ) of bacteria and archaea is essential for this tRNA to read the isoleucine codon AUA and to differentiate between AUA and the methionine codon AUG. To identify the modified cytidine in archaea, we have purified this tRNA species from Haloarcula marismortui, established its codon reading properties, used liquid chromatography-mass spectrometry (LC-MS) to map RNase A and T1 digestion products onto the tRNA, and used LC-MS/MS to sequence the oligonucleotides in RNase A digests. These analyses revealed that the modification of cytidine in the anticodon of tRNA Ile 2 adds 112 mass units to its molecular mass and makes the glycosidic bond unusually labile during mass spectral analyses. Accurate mass LC-MS and LC-MS/MS analysis of total nucleoside digests of the tRNA Ile 2 demonstrated the absence in the modified cytidine of the C2-oxo group and its replacement by agmatine (decarboxy-arginine) through a secondary amine linkage. We propose the name agmatidine, abbreviation C þ , for this modified cytidine. Agmatidine is also present in Methanococcus maripaludis tRNA Ile 2 and in Sulfolobus solfataricus total tRNA, indicating its probable occurrence in the AUA decoding tRNA Ile of euryarchaea and crenarchaea. The identification of agmatidine shows that bacteria and archaea have developed very similar strategies for reading the isoleucine codon AUA while discriminating against the methionine codon AUG.agmatine | decoding | RNA modification | tRNA | wobble pairing T he genetic code table consists of sixteen four-codon boxes. In fourteen of the boxes, all four codons either specify the same amino acid or are split into two sets of two codons, with each set encoding a different amino acid. For example, the UUN box is split into UUU/UUC coding for phenylalanine and UUA/UUG coding for leucine. The wobble hypothesis of Crick proposes how a single phenylalanine tRNA with G in the first anticodon position can base pair with either U or C and a single leucine tRNA with a modified U (or 2-thioU) in the anticodon can base pair with either A or G (1-3). The two remaining boxes, UGN and AUN, are exceptions in that the UGN box is split into UGU/UGC coding for cysteine, UGG coding for tryptophan, and UGA being used as a stop codon, whereas the AUN box is split into AUU/AUC/AUA coding for isoleucine and AUG coding for methionine. The isoleucine codons AUU and AUC can be read by an isoleucine tRNA with G in the anticodon following the wobble pairing rules, but how the AUA codon is read specifically by a tRNA Ile without also reading the AUG codon has been a question of much interest over the years.Different organisms have developed different strategies for reading the AUA codon. Bacteria use a tRNA Ile with the anticodon LAU (L ¼ lysidine) (4-7). Lysidine is a modified cytidine in which the C2-oxo group of cytidine is replaced by lysine. Exactly how it base pairs with A but not with G is not established.Eukaryotes, on the other hand, con...

Transfer RNAs (tRNA) through their abundance and modification pattern significantly influence protein translation. Here, we present a systematic analysis of the tRNAome of Lactococcus lactis. Using the next-generation sequencing approach, we identified 40 tRNAs which carry 16 different posttranscriptional modifications as revealed by mass spectrometry analysis. While small modifications are located in the tRNA body, hypermodified nucleotides are mainly present in the anticodon loop, which through wobbling expand the decoding potential of the tRNAs. Using tRNA-based microarrays, we also determined the dynamics in tRNA abundance upon changes in the growth rate and heterologous protein overexpression stress. With a four-fold increase in the growth rate, the relative abundance of tRNAs cognate to low abundance codons decrease, while the tRNAs cognate to major codons remain mostly unchanged. Significant changes in the tRNA abundances are observed upon protein overexpression stress, which does not correlate with the codon usage of the overexpressed gene but rather reflects the altered expression of housekeeping genes.

Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. The functional dynamics, synergistic nature and regulatory roles of these posttranscriptional nucleoside modifications within the cell are not well characterized. These modifications are present at very low levels and isolation of individual nucleosides for analysis requires a complex multi-step approach. The focus of this study is to characterize the reproducibility of a liquid chromatography method used to isolate and quantitatively characterize modified nucleosides in tRNA and rRNA when nucleoside detection is performed using ultraviolet and mass spectrometric detection (UV and MS, respectively). Despite the analytical challenges of sample isolation and dynamic range, quantitative profiling of modified nucleosides obtained from bacterial tRNAs and rRNAs is feasible at relative standard deviations of 5% RSD or less.

Methylthiotransferases (MTTases) are a closely related family of proteins that perform both radical-S-adenosylmethionine (SAM) mediated sulfur insertion and SAM-dependent methylation to modify nucleic acid or protein targets with a methyl thioether group (–SCH3). Members of two of the four known subgroups of MTTases have been characterized, typified by MiaB, which modifies N6-isopentenyladenosine (i6A) to 2-methylthio-N6-isopentenyladenosine (ms2i6A) in tRNA, and RimO, which modifies a specific aspartate residue in ribosomal protein S12. In this work, we have characterized the two MTTases encoded by Bacillus subtilis 168 and find that, consistent with bioinformatic predictions, ymcB is required for ms2i6A formation (MiaB activity), and yqeV is required for modification of N6-threonylcarbamoyladenosine (t6A) to 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in tRNA. The enzyme responsible for the latter activity belongs to a third MTTase subgroup, no member of which has previously been characterized. We performed domain-swapping experiments between YmcB and YqeV to narrow down the protein domain(s) responsible for distinguishing i6A from t6A and found that the C-terminal TRAM domain, putatively involved with RNA binding, is likely not involved with this discrimination. Finally, we performed a computational analysis to identify candidate residues outside the TRAM domain that may be involved with substrate recognition. These residues represent interesting targets for further analysis.

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