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Expanded Use of Sense Codons Is Regulated by Modified Cytidines in Trna

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Expanded Use of Sense Codons Is Regulated by Modified Cytidines in Trna Expanded use of sense codons is regulated by modified cytidines in tRNA William A. Cantaraa,b,1, Frank V. Murphy IVc, Hasan Demircid,2, and Paul F. Agrisa,3 aThe RNA Institute, Department of Biological Sciences, University at Albany–State University of New York, Albany, NY 12222; bDepartment of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622; cNortheastern Collaborative Access Team, Argonne National Laboratory, Argonne, IL 60439; and dDepartment of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912 Edited by Dieter Söll, Yale University, New Haven, CT, and approved May 23, 2013 (received for review December 26, 2012) Codon use among the three domains of life is not confined to the C-H edge at the C5 position. It is evident that the many post- universal genetic code. With only 22 tRNA genes in mammalian transcriptional modifications of the Watson–Crick edge alter base mitochondria, exceptions from the universal code are necessary pairing abilities, but a clear mechanism of decoding expansion for proper translation. A particularly interesting deviation is the by C5 modifications remains poorly understood, especially mod- decoding of the isoleucine AUA codon as methionine by the one fi Met i cations of cytidine. mitochondrial-encoded tRNA . This tRNA decodes AUA and AUG The mitochondrion’s decoding of AUA as methionine is in both the A- and P-sites of the metazoan mitochondrial ribo- important for proper translation. In humans, this codon con- some. Enrichment of posttranscriptional modifications is a com- monly appropriated mechanism for modulating decoding rules, stitutes 20% of mRNA initiator methionines and 80% of in- enabling some tRNA functions while restraining others. In this ternal methionines (15, 16). Using chemical synthesis and 5 5 fi case, a modification of cytidine, 5-formylcytidine (f C), at the wob- incorporation of the f C34 modi cation into the heptadecamer Met Met ble position-34 of human mitochondrial tRNAf5CAU (hmtRNAf5CAU) anticodon stem and loop domain (ASL) of human mitochon- Met Met 5 enables expanded decoding of AUA, resulting in a deviation in the drial tRNA ðhmASL Þ (17), we determined that f C34 • f5CAU f5CAU genetic code. Visualization of the codon anticodon interaction by destabilized the hmASLMet by increasing the motional dy- X-ray crystallography revealed that recognition of both A and G at f5CAU the third position of the codon occurs in the canonical Watson– namics of the loop residues (17). A further study detailing Crick geometry. A modification-dependent shift in the tautomeric the codon-binding characteristics and solution structure of Met 5 equilibrium toward the rare imino-oxo tautomer of cytidine stabil- hmASLf5CAU agreed with the f C-dependent increase in resi- 5 • izes the f C34 A base pair geometry with two hydrogen bonds. due dynamics (18). Proposed mechanisms for the decoding Met 5 • of AUA by tRNAf5CAU depend on the f C34 A3 base pair modified nucleosides | ribosome crystallography | tautomerism forming a specificgeometry(18–20). Based on molecular dynamics simulations, we suggested that this base pair could he genetic code was initially deemed to be universal and frozen be in a sheared geometry that is supported by a bridging water Tin time (1). However, deviations in sense and nonsense codon molecule (18). To test this hypothesis, the geometry of the use are found in bacteria, archaea, and both nuclear and organ- f5C •A base pair in the decoding center of the ribosomal ellar eukaryotic genomes (2, 3). Use of genetic codes that deviate 34 3 A-site was observed in crystal structures of natively modified from the universal code provides insight into its evolution (4) and Met fi possibilities for investigator-initiated manipulation (synthetic hmASLf5CAU bound to AUA. Here, we show for the rst time fi 5 • biology) (5). In many cases, however, the translation of the de- the modi cation-dependent f C34 A3 base pair within the viant sense codons is facilitated by posttranscriptional modifi- codon•anticodon interaction during ribosomal A-site decod- 5 cation chemistries that are enzymatically added to nucleosides ing. Surprisingly, f C34 forms a canonical Watson–Crick base at the first anticodon position. The modification chemistries and pair with both the G of AUG and the A of AUA, refuting the their impact on anticodon conformation alter the decoding ca- conformation predicted from molecular dynamics simulations pacity of the modified tRNA (6). When first proposed, modifi- 5 (18). This geometry of the f C34•A3 base pair requires a novel cation-dependent wobble decoding was limited to inosine as the 5 amino-imino tautomerism in f C34 similar to the keto-enol tau- first modified anticodon residue. Inosine, a deaminated adeno- 5 tomerism seen for the two wobble position uridines cmo U34 sine residue, expands the ability of a single isoacceptor tRNA to 5 2 Val3 read three codons by base pairing with either U, C, or A at the and mcm s U34 in Escherichia coli tRNAUAC (21) and human – Lys3 third position of the codon (7). Many other wobble position tRNAUUU (22), respectively. modified residues, mostly pyrimidines, are now known to modulate use of specific codons (8). Although modified uridines constitute a great majority of these modifications, modified cytidines are prevalent in controlling a switch between the universal genetic Author contributions: P.F.A. designed research; W.A.C., F.V.M., and H.D. performed re- code and a deviant code in the shared isoleucine/methionine search; W.A.C., F.V.M., H.D., and P.F.A. analyzed data; and W.A.C., F.V.M., H.D., and P.F.A. fi 4 wrote the paper. codon box (9, 10). The modi cation, 4-acetylcytidine (ac C), fl Met The authors declare no con ict of interest. prevents tRNACAU from reading AUA through wobble geometry (11), and lysidine (k2C) (12) and agmatidine (agm2C) (13, 14) This article is a PNAS Direct Submission. prevent tRNAIle from reading AUG in bacteria and archaea. Freely available online through the PNAS open access option. CAU Met Met Interestingly, a fourth cytidine modification, 5-formylcytidine Data deposition: The atomic coordinates for hmASL f5CAU-AUG and hmASL f5CAU-AUA 5 have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4GKJ and (f C), facilitates the reading of AUA and AUG as methionine by 4GKK, respectively). Met a single tRNAf5CAU responding to both initiator and elongator 1Present address: Department of Chemistry and Biochemistry, Center for Retroviral Re- codons in yeast and many metazoan mitochondrial genomes (6). search, and Center for RNA Biology, Ohio State University, Columbus, OH 43210. 4 2 2 Therefore, ac C, k C and agm C are restrictive modifications 2Present address: Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo that alter the physicochemical properties of the Watson–Crick Park, CA 94025. 5 edge, whereas f C expands decoding using a modification on the 3To whom correspondence should be addressed. E-mail: [email protected]. 10964–10969 | PNAS | July 2, 2013 | vol. 110 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1222641110 Downloaded by guest on September 29, 2021 Met • Fig. 1. hmASLf5CAU secondary structure and A-site codon anticodon interaction. (A) The ASL was synthesized as a heptadecamer containing two mod- fi Met i cations: pseudouridine at position 27 and 5-formylcytidine at position 34. (B) The structure of hmASLf5CAU bound to AUG (Left) showed strong electron density for ASL residues 31–39, whereas when bound to AUA (Right), electron density was strong for only residues 34–39. In both structures, the ASL is in 5 green, the mRNA codon is in blue, and the A-site interacting residues (G530,A1492, and A1493) are in black (2mFO-dFC contoured at 1.5 σ). The f C34 modification is colored red. Results structures. The structure of the 30S ribosomal subunit (including hmASLMet all RNA and protein) was nearly identical to those reported Codon Bound f5CAU Structure. The ASL domain of Met previously (21, 22). The hmASLMet took a conformation hmtRNAf5CAU was chemically synthesized with the wobble f5CAU 5 modification f C34, the native C33, and a pseudouridine, Ψ27,at nearly identical to that of the ASL of a ribosome-bound tRNA- the 5′ terminus (Fig. 1A). C33 is quite rare. The uridine at po- EF-Tu complex (24, 25), demonstrating the biological relevance sition 33 in tRNAs is considered invariant and recognized for its of the present structures. More importantly, the conserved A-site contribution to the “U-turn” structural motif. There are only 21 residues G530,A1492, and A1493 were in the correct orientation to • known instances of C33, 13 of which are found in initiator tRNAs constrain the codon anticodon pair residues into the proper (23). The hmASLMet bound both AUG and AUA codons with geometry for recognition (Fig. 1B) (26, 27). As such, differences f5CAU • significant affinity in the A-site of the bacterial ribosome (17) in the characteristics of the codon anticodon interaction can fi Met and within the A-site of the bovine mitochondrial 55S ribosome be attributed to the speci cconformationofhmASLf5CAU and 5 fi (18). In determining the crystallographic structure of the modi- properties of the f C34 modi cation. fi Met ed hmASLf5CAU bound to the AUG or the AUA codons in the hmASLMet : Conformational Characteristics of f5CAU Nearly the entire ribosomal A-site, native Thermus thermophilus 30S ribosomal Met Met hmASLf5CAU exhibited strong electron density when bound to the subunit crystals were soaked with the hmASLf5CAU and hex- Met americ oligonucleotides, each containing either AUG or AUA cognate AUG codon (Fig. 1B). In the structure of hmASLf5CAU • codons. Under the conditions used, both crystals diffracted bound to the wobble AUA codon, the codon anticodon in- ′ to 3.3-Å resolution (Table 1).
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