Agmatidine, a modified cytidine in the anticodon of archaeal tRNA ^Ile , base pairs with adenosine but not with guanosine

Abstract

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...

Celebrating wobble decoding: Half a century and still much is new

“…As an example, isoleucine shares a codon box with methionine where AUA, AUC, and AUU code for isoleucine, and AUG codes for methionine, with the exception of mitochondria where AUA also codes for methionine. 6,113,122 Two codons can be read by tRNA Ile GAU , but the AUA codon requires a different isoacceptor that would also not read AUG as isoleucine. Eukaryotes commonly have a tRNA Ile IAU which can read all three codons, but not AUG. 122 The genomes of bacteria and some eukaryotic organelles encode a tRNA Ile GAU and tRNA Ile LAU where L is lysidine (k 2 C).…”

“…6,113,122 Two codons can be read by tRNA Ile GAU , but the AUA codon requires a different isoacceptor that would also not read AUG as isoleucine. Eukaryotes commonly have a tRNA Ile IAU which can read all three codons, but not AUG. 122 The genomes of bacteria and some eukaryotic organelles encode a tRNA Ile GAU and tRNA Ile LAU where L is lysidine (k 2 C). 123 Modification of C to k 2 C (Fig.…”

“…Thus, there have been multiple and convergent evolutionary paths to accomplish specificity in decoding through restriction of codon recognition with the help of modified nucleosides. 122

10.1080/15476286.2017.1356562-f0007Figure 7.Modifications influence protein and codon recognition of tRNA through altered hydrogen bonding. R represents the ribose sugar.

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“…The exact tautomeric form through which cognate codon recognition can occur still needs to be elucidated. 122 However, it is a tautomer of lysidine that disallows recognition of the near-cognate AUG codon. The modification contains a secondary amine in place of oxygen at the two position of C, thus switching a hydrogen bond acceptor into a donor.…”

“…126 Agmatidine forms a similar tautomer as lysidine. 122,126 The hydrogen bonding capability of modified nucleosides commonly found at the wobble position of tRNA Ile illustrates how small changes in the chemistry of a single nucleoside can have broad and highly specific effects, in this case altering codon recognition and specificity while simultaneously preserving aaRS recognition. 121 …”