@article{cantara_murphy_demirci_agris_2013, title={Expanded use of sense codons is regulated by modified cytidines in tRNA}, volume={110}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1222641110}, abstractNote={ Codon use among the three domains of life is not confined to the universal genetic code. With only 22 tRNA genes in mammalian mitochondria, exceptions from the universal code are necessary for proper translation. A particularly interesting deviation is the decoding of the isoleucine AUA codon as methionine by the one mitochondrial-encoded tRNA Met . This tRNA decodes AUA and AUG in both the A- and P-sites of the metazoan mitochondrial ribosome. Enrichment of posttranscriptional modifications is a commonly appropriated mechanism for modulating decoding rules, enabling some tRNA functions while restraining others. In this case, a modification of cytidine, 5-formylcytidine (f 5 C), at the wobble position-34 of human mitochondrial ( ) enables expanded decoding of AUA, resulting in a deviation in the genetic code. Visualization of the codon•anticodon interaction by X-ray crystallography revealed that recognition of both A and G at the third position of the codon occurs in the canonical Watson–Crick geometry. A modification-dependent shift in the tautomeric equilibrium toward the rare imino-oxo tautomer of cytidine stabilizes the f 5 C 34 •A base pair geometry with two hydrogen bonds. }, number={27}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Cantara, William A. and Murphy, Frank V. and Demirci, Hasan and Agris, Paul F.}, year={2013}, month={Jul}, pages={10964–10969} } @article{vendeix_murphy_cantara_leszczynska_gustilo_sproat_malkiewicz_agris_2012, title={Human tRNA(UUU)(LYs3) Is Pre-Structured by Natural Modifications for Cognate and Wobble Codon Binding through Keto-Enol Tautomerism}, volume={416}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2011.12.048}, abstractNote={Human tRNA(Lys3)(UUU) (htRNA(Lys3)(UUU)) decodes the lysine codons AAA and AAG during translation and also plays a crucial role as the primer for HIV-1 (human immunodeficiency virus type 1) reverse transcription. The posttranscriptional modifications 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U(34)), 2-methylthio-N(6)-threonylcarbamoyladenosine (ms(2)t(6)A(37)), and pseudouridine (Ψ(39)) in the tRNA's anticodon domain are critical for ribosomal binding and HIV-1 reverse transcription. To understand the importance of modified nucleoside contributions, we determined the structure and function of this tRNA's anticodon stem and loop (ASL) domain with these modifications at positions 34, 37, and 39, respectively (hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39)). Ribosome binding assays in vitro revealed that the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) bound AAA and AAG codons, whereas binding of the unmodified ASL(Lys3)(UUU) was barely detectable. The UV hyperchromicity, the circular dichroism, and the structural analyses indicated that Ψ(39) enhanced the thermodynamic stability of the ASL through base stacking while ms(2)t(6)A(37) restrained the anticodon to adopt an open loop conformation that is required for ribosomal binding. The NMR-restrained molecular-dynamics-derived solution structure revealed that the modifications provided an open, ordered loop for codon binding. The crystal structures of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) bound to the 30S ribosomal subunit with each codon in the A site showed that the modified nucleotides mcm(5)s(2)U(34) and ms(2)t(6)A(37) participate in the stability of the anticodon-codon interaction. Importantly, the mcm(5)s(2)U(34)·G(3) wobble base pair is in the Watson-Crick geometry, requiring unusual hydrogen bonding to G in which mcm(5)s(2)U(34) must shift from the keto to the enol form. The results unambiguously demonstrate that modifications pre-structure the anticodon as a key prerequisite for efficient and accurate recognition of cognate and wobble codons.}, number={4}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Vendeix, Franck A. P. and Murphy, Frank V. and Cantara, William A. and Leszczynska, Grazyna and Gustilo, Estella M. and Sproat, Brian and Malkiewicz, Andrzej and Agris, Paul F.}, year={2012}, month={Mar}, pages={467–485} } @article{cantara_bilbille_kim_kaiser_leszczynska_malkiewicz_agris_2012, title={Modifications Modulate Anticodon Loop Dynamics and Codon Recognition of E. coli tRNA (Arg1,2)}, volume={416}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2011.12.054}, abstractNote={Three of six arginine codons are read by two tRNAArg isoacceptors in Escherichia coli. The anticodon stem and loop of these isoacceptors (ASLArg1,2) differs only in that the position 32 cytidine of tRNAArg1 is posttranscriptionally modified to 2-thiocytidine (s2C32). The tRNAArg1,2 are also modified at positions 34 (inosine, I34) and 37 (2-methyladenosine, m2A37). To investigate the roles of modifications in the structure and function, we analyzed six ASLArg1,2 constructs differing in their array of modifications by spectroscopy and codon binding assays. Thermal denaturation and circular dichroism spectroscopy indicated that modifications contribute thermodynamic and base stacking properties, resulting in more order but less stability. NMR-derived structures of the ASLArg1,2 showed that the solution structures of the ASLs were nearly identical. Surprisingly, none possessed the U-turn conformation required for effective codon binding on the ribosome. Yet, all ASLArg1,2 constructs efficiently bound the cognate CGU codon. Three ASLs with I34 were able to decode CGC, whereas only the singly modified ASLArg1,2ICG with I34 was able to decode CGA. The dissociation constants for all codon bindings were physiologically relevant (0.4–1.4 μM). However, with the introduction of s2C32 or m2A37 to ASLArg1,2ICG, the maximum amount of ASL bound to CGU and CGC was significantly reduced. These results suggest that, by allowing loop flexibility, the modifications modulate the conformation of the ASLArg1,2, which takes one structure free in solution and two others when bound to the cognate arginyl-tRNA synthetase or to codons on the ribosome where modifications reduce or restrict binding to specific codons.}, number={4}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Cantara, William A. and Bilbille, Yann and Kim, Jia and Kaiser, Rob and Leszczynska, Grazyna and Malkiewicz, Andrzej and Agris, Paul F.}, year={2012}, month={Mar}, pages={579–597} } @article{lusic_gustilo_vendeix_kaiser_delaney_graham_moye_cantara_agris_deiters_2008, title={Synthesis and investigation of the 5-formylcytidine modified, anticodon stem and loop of the human mitochondrial tRNA(Met)}, volume={36}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkn703}, abstractNote={Human mitochondrial methionine transfer RNA (hmtRNAMetCAU) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position-34 (f5C34). The role of this modification in (hmtRNAMetCAU) for the decoding of AUA, as well as AUG, in both the peptidyl- and aminoacyl-sites of the ribosome in either chain initiation or chain elongation is still unknown. We report the first synthesis and analyses of the tRNA's anticodon stem and loop domain containing the 5-formylcytidine modification. The modification contributes to the tRNA's anticodon domain structure, thermodynamic properties and its ability to bind codons AUA and AUG in translational initiation and elongation.}, number={20}, journal={NUCLEIC ACIDS RESEARCH}, author={Lusic, Hrvoje and Gustilo, Estella M. and Vendeix, Franck A. P. and Kaiser, Rob and Delaney, Michael O. and Graham, William D. and Moye, Virginia A. and Cantara, William A. and Agris, Paul F. and Deiters, Alexander}, year={2008}, month={Nov}, pages={6548–6557} }