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Agmatidine, a Modified Cytidine in the Anticodon of Archaeal Trna , Base

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Agmatidine, a Modified Cytidine in the Anticodon of Archaeal Trna , Base Agmatidine, a modified cytidine in the anticodon of archaeal tRNAIle, base pairs with adenosine but not with guanosine Debabrata Mandala,1, Caroline Köhrera,1, Dan Sub, Susan P. Russellc, Kady Krivosc, Colette M. Castleberryc, Paul Blumd, Patrick A. Limbachc, Dieter Söllb, and Uttam L. RajBhandarya,2 aDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; bDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520; cDepartment of Chemistry, University of Cincinnati, Cincinnati, OH 45221; dGeorge Beadle Center for Genetics, University of Nebraska, Lincoln, NE 68588 Contributed by Dieter Söll, December 24, 2009 (sent for review December 2, 2009) Modification of the cytidine in the first anticodon position of the Eukaryotes, on the other hand, contain a tRNAIle with the anti- Ile Ile AUA decoding tRNA (tRNA2 ) of bacteria and archaea is essential codon IAU (I ¼ inosine), which can read all three isoleucine co- for this tRNA to read the isoleucine codon AUA and to differentiate dons using the wobble pairing rules of Crick. They also contain a between AUA and the methionine codon AUG. To identify the tRNAIle with the anticodon ΨAΨ, which is thought to read only modified cytidine in archaea, we have purified this tRNA species the isoleucine codon AUA but not the methionine codon AUG from Haloarcula marismortui, established its codon reading proper- (8). Given these two distinct mechanisms in bacteria and ties, used liquid chromatography–mass spectrometry (LC-MS) to eukaryotes, a question of much interest is how the archaeal map RNase A and T1 digestion products onto the tRNA, and used tRNAIle accomplishes the task of reading the AUA codon. LC-MS/MS to sequence the oligonucleotides in RNase A digests. In a recent paper, we showed that a tRNA with the anticodon These analyses revealed that the modification of cytidine in the an- sequence CAU, which was annotated as a methionine tRNA in Ile ticodon of tRNA2 adds 112 mass units to its molecular mass and the archaeon Haloarcula marismortui, was actually aminoacylated makes the glycosidic bond unusually labile during mass spectral with isoleucine in vivo (9). We showed that the cytidine in the analyses. Accurate mass LC-MS and LC-MS/MS analysis of total anticodon of this tRNA was modified, but not to lysidine as it Ile nucleoside digests of the tRNA2 demonstrated the absence in is in bacteria, and that the same modification was likely present the modified cytidine of the C2-oxo group and its replacement in other haloarchaea and in Methanocaldococcus jannaschii.We by agmatine (decarboxy-arginine) through a secondary amine also showed that modification of the cytidine was necessary for linkage. We propose the name agmatidine, abbreviation Cþ, for aminoacylation of the tRNA with isoleucine in vitro. These find- this modified cytidine. Agmatidine is also present in Methanococ- ings raised the question of the nature of the modification in the cus maripaludis Ile Sulfolobus solfataricus Ile Ile tRNA2 and in total tRNA, archaeal AUA decoding tRNA (tRNA2 ) and its relationship, Ile Ile indicating its probable occurrence in the AUA decoding tRNA if any, to lysidine in Escherichia coli tRNA2 . of euryarchaea and crenarchaea. The identification of agmatidine Here, we describe the purification of two tRNAIle species from Ile shows that bacteria and archaea have developed very similar stra- H. marismortui. We show that tRNA1 binds to AUC but not to Ile tegies for reading the isoleucine codon AUA while discriminating AUA or AUG on the ribosome, whereas tRNA2 binds to AUA against the methionine codon AUG. but not to AUC, AUG, or AUU. Mass spectral analysis of nucleo- Ile sides and oligonucleotides isolated from tRNA2 show that the agmatine ∣ decoding ∣ RNA modification ∣ tRNA ∣ wobble pairing modified cytidine is located in the anticodon wobble position, lacks the C2-oxo group, and has instead agmatine (1-amino he genetic code table consists of sixteen four-codon boxes. 4-guanidino butane) attached to it through a secondary amine TIn fourteen of the boxes, all four codons either specify the linkage. We propose the name agmatidine and the abbreviation same amino acid or are split into two sets of two codons, with Cþ for this modified nucleoside. Agmatidine is also present in Ile each set encoding a different amino acid. For example, the tRNA2 from Methanococcus maripaludis and in total tRNA iso- UUN box is split into UUU/UUC coding for phenylalanine lated from Sulfolobus solfataricus, indicating its possible presence Ile and UUA/UUG coding for leucine. The wobble hypothesis of in tRNA2 of euryarchaea and crenarchaea. Crick proposes how a single phenylalanine tRNA with G in Agmatidine is in many ways similar to lysidine. In both cases the first anticodon position can base pair with either U or C the C2-oxo group of cytidine is replaced by either of two closely and a single leucine tRNA with a modified U (or 2-thioU) in related basic amino acids, decarboxy-arginine or lysine. Thus, the anticodon can base pair with either A or G (1–3). The two bacteria and archaea have developed very similar strategies for Ile remaining boxes, UGN and AUN, are exceptions in that the generating a tRNA2 to read the isoleucine codon AUA without UGN box is split into UGU/UGC coding for cysteine, UGG also reading the methionine codon AUG. The identification of coding for tryptophan, and UGA being used as a stop codon, agmatidine also suggests that agmatine, which is a known whereas the AUN box is split into AUU/AUC/AUA coding for isoleucine and AUG coding for methionine. The isoleucine co- Author contributions: D.M., C.K., P.A.L., D. Söll, and U.L.R. designed research; D.M., C.K., dons AUU and AUC can be read by an isoleucine tRNA with D. Su, S.P.R., K.K., and C.M.C. performed research; K.K. and P.B. contributed new reagents/ G in the anticodon following the wobble pairing rules, but analytic tools; D.M., C.K., S.P.R., K.K., C.M.C., P.A.L., D. Söll, and U.L.R. analyzed data; and how the AUA codon is read specifically by a tRNAIle without also C.K., P.A.L., D. Söll, and U.L.R. wrote the paper. reading the AUG codon has been a question of much interest The authors declare no conflict of interest. over the years. Freely available online through the PNAS open access option. Different organisms have developed different strategies for 1D.M. and C.K. contributed equally to this work. Ile reading the AUA codon. Bacteria use a tRNA with the antic- 2To whom correspondence should be addressed at: Department of Biology, Room 68-671, odon LAU (L ¼ lysidine) (4–7). Lysidine is a modified cytidine in M.I.T., Cambridge, MA 02139. E-mail: [email protected]. which the C2-oxo group of cytidine is replaced by lysine. Exactly This article contains supporting information online at www.pnas.org/cgi/content/full/ how it base pairs with A but not with G is not established. 0914869107/DCSupplemental. 2872–2877 ∣ PNAS ∣ February 16, 2010 ∣ vol. 107 ∣ no. 7 www.pnas.org/cgi/doi/10.1073/pnas.0914869107 Downloaded by guest on September 26, 2021 neuromodulator (10, 11), and which is essential for polyamine of the phosphodiester bond on the 3′-side of the dimethyl-G biosynthesis in the archaeon Thermococcus kodakaraensis (12), residue (15). may also be essential in euryarchaea and in crenarchaea (13) The pattern obtained in partial alkali digests shows a pro- 32 for modification of the cytidine required for decoding the nounced shift in gel electrophoretic mobility of the 5′- P-labeled Ile AUA codon by tRNA2 . oligonucleotide going from nucleotide 33 to 34. We had shown previously that nucleotide 34 contained a modified cytidine Results (9). The presence of nine bands between G31 and G41 in the Purification and Characterization of Isoleucine tRNAs from H. maris- alkali digest shows that every phosphodiester bond between mortui. Starting from total tRNA, a two-step procedure, involving G31 and G41 is cleaved by alkali. This means that none of the hybrid selection of tRNA with biotinylated DNA oligonucleo- nucleotides in between contain a substitution in the 2′-hydroxyl tides bound to streptavidin sepharose followed by native polya- group. Therefore, the very pronounced shift in the gel electro- crylamide gel fractionation of the enriched tRNA, was used to phoretic mobility indicates strongly that the modified cytidine isolate pure tRNAIles. The biotinylated oligonucleotides were has at least one if not more positive charges in the ring, which Ile Ile complementary to nucleotides 54–73 of tRNA1 or tRNA2 would retard the electrophoretic migration of the oligonucleotide (Fig. 1A) and contained biotin at the 3′-hydroxyl end. The final that contained the modified cytidine (16). yields of purified tRNAs from approximately 1; 000 A260 units of Ile Ile Ile Ile Codon Reading Properties of tRNA and tRNA . 6–8 260 0 6–0 8 260 1 2 Purified tRNA1 total tRNA were A units for tRNA1 and . A unit Ile 3 Ile Ile and tRNA2 were aminoacylated in vitro with H-Ile and the for tRNA2 . The significantly lower yield of tRNA2 compared to 3 Ile H-Ile-tRNAs were used for measuring oligonucleotide-directed tRNA1 reflects the low abundance of this tRNA and the fact H. marismortui that AUA is a rarely used codon in haloarchaea as it is in E. coli binding to ribosomes. The oligonucleotides used (14). as template had the following sequences AUGAUC, AUGAUA, Fig. 1B shows that the purified tRNAs are essentially homo- AUGAUG and AUGAUU. Results of ribosome binding experiments show that tRNAIle binds to AUC but not to AUA geneous.
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