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) (tRNA2(Ile)) 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 tRNA2(Ile) 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 tRNA2(Ile) 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(+), for this modified cytidine. Agmatidine is also present in Methanococcus maripaludis tRNA2(Ile) 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.

Fig. 1.

(A) Cloverleaf structure of from H. marismortui. Location of modified nucleosides is based on Gupta (46) and LC-MS analysis presented in this work. G⁺ (archaeosine); (N², N²-dimethylguanosine); C* (modified cytidine); t⁶A (N⁶-threonylcarbamoyladenosine); m⁵C (5-methylcytidine); m¹ψ (1-methylpseudouridine); ψ (pseudouridine); C_m (2′-O-methylcytidine); m¹I (1-methylinosine). (B) Purification of isoleucine tRNAs from H. marismortui. Native PAGE analysis of total tRNA, purified (Ile2) and (Ile1) is shown. tRNAs are visualized by ethidium bromide staining or Northern blot analysis using probes specific for and as indicated. (C) Characterization of purified . The homogeneity of was confirmed by partial RNase T1 digest (lane T1) of 5′-³²P-labeled tRNA. ³²P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography; lane OH^-, partial alkali digest.

Fig. 2.

Ribosome binding of H. marismortui and . Template-dependent binding of purified (A) and (B) to ribosomes isolated from H. marismortui. Oligonucleotides used were AUGAUG (▵), AUGAUA (□) and AUGAUC (○).

Fig. 3.

LC-MS/MS analysis of oligonucleotides present in RNase A/BAP digests of H. marismortui . (A) TIC of RNase A/BAP digestion of H. marismortui . (B) SIC of the anticodon-derived trinucleoside diphosphate 5′-C*pApU-3′ at m/z 989.3. (C) Mass spectrum of oligonucleotides eluting at 4.5 min. (D) MS/MS analysis of the trinucleoside diphosphate at m/z 989.3 (22).

Fig. 4.

Mass spectral analysis of the modified cytidine C^∗. Purified H. marismortui was digested to nucleosides and analyzed by LC-UV-MS (for complete analysis see Fig. S3). Selected ion chromatograms of (A) the molecular ion at m/z 356 (MH⁺) and (B) the base ion at m/z 224 (). (C) Mass spectrum of the modified cytidine C*; C* coelutes with the leading edge of archaeosine.

Fig. 5.

Identification of the modified cytidine C*. (A) Accurate mass analysis of CID-MS/MS fragments of the base ion of C* (measured m/z 224.1617; expected m/z 224.1618). m/z 207.1352 and m/z 165.1135 are consistent with the loss of NH₃ and CH₅N₃, respectively, from the base ion of C*. (B) MS fragmentation pattern of an agmatine standard. m/z 114.1 and m/z 72.1 are consistent with the loss of NH₃ and CH₅N₃, respectively, from agmatine.

Fig. 6.

Comparison of agmatidine present in the anticodon wobble position of the archaeal AUA-decoding to lysidine present in the corresponding bacterial tRNA. Possible tautomeric structures of protonated agmatidine and lysidine are shown. Tautomers 1 and 2 could base pair with A.