@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{vendeix_dziergowska_gustilo_graham_sproat_malkiewicz_agris_2008, title={Anticodon domain modifications contribute order to tRNA for ribosome-mediated codon binding}, volume={47}, ISSN={["0006-2960"]}, DOI={10.1021/bi702356j}, abstractNote={The accuracy and efficiency with which tRNA decodes genomic information into proteins require posttranscriptional modifications in or adjacent to the anticodon. The modification uridine-5-oxyacetic acid (cmo (5)U 34) is found at wobble position 34 in a single isoaccepting tRNA species for six amino acids, alanine, leucine, proline, serine, threonine, and valine, each having 4-fold degenerate codons. cmo (5)U 34 makes possible the decoding of 24 codons by just six tRNAs. The contributions of this important modification to the structures and codon binding affinities of the unmodified and fully modified anticodon stem and loop domains of tRNA (Val3) UAC (ASL (Val3) UAC) were elucidated. The stems of the unmodified ASL (Val3) UAC and that with cmo (5)U 34 and N (6)-methyladenosine, m (6)A 37, adopted an A-form RNA conformation (rmsd approximately 0.6 A) as determined with NMR spectroscopy and torsion-angle molecular dynamics. However, the UV hyperchromicity, circular dichroism ellipticity, and structural analyses indicated that the anticodon modifications enhanced order in the loop. ASL (Val3) UAC-cmo (5)U 34;m (6)A 37 exhibited high affinities for its cognate and wobble codons GUA and GUG, and for GUU in the A-site of the programmed 30S ribosomal subunit, whereas the unmodified ASL (Val3) UAC bound less strongly to GUA and not at all to GUG and GUU. Together with recent crystal structures of ASL (Val3) UAC-cmo (5)U 34;m (6)A 37 bound to all four of the valine codons in the A-site of the ribosome's 30S subunit, these results clearly demonstrate that the xo (5)U 34-type modifications order the anticodon loop prior to A-site codon binding for an expanded codon reading, possibly reducing an entropic energy barrier to codon binding.}, number={23}, journal={BIOCHEMISTRY}, author={Vendeix, Franck A. P. and Dziergowska, Agnieszka and Gustilo, Estella M. and Graham, William D. and Sproat, Brian and Malkiewicz, Andrzej and Agris, Paul F.}, year={2008}, month={Jun}, pages={6117–6129} }
 @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} }
 @misc{gustilo_franck_agris_2008, title={tRNA's modifications bring order to gene expression}, volume={11}, number={2}, journal={Current Opinion in Microbiology}, author={Gustilo, E. M. and Franck, A. P. F. and Agris, P. F.}, year={2008}, pages={134–140} }
 @article{gustilo_dubois_lapointe_agris_2007, title={E-coli glutamyl-tRNA synthetase is inhibited by anticodon stem-loop domains and a minihelix}, volume={4}, ISSN={["1555-8584"]}, DOI={10.4161/rna.4.2.4736}, abstractNote={Portions of E. coli tRNAGlu having identity determinants for glutamyl-tRNA synthetase (ERS, EC 6.1.1.17) have been designed to be the first RNA inhibitors of a Class I synthetase. ERS recognizes the 2-thionyl group of 2-thio-5-methylaminomethyluridine (mnm5s2U34) in the first or wobble anticodon position of E. coli tRNAGlu. The interaction, as revealed by structural analysis, though specific, appears tenuous. Thus, it is surprising that RNAs designed from this tRNA's anticodon stem and loop domain with (ASLGlu-s2U34) and without s2U34 are bound by ERS and inhibit aminoacylation of the native tRNA. ASLGlu, ASLGlu-s2U34, and a minihelixGlu composed of identity determinants of the amino acid accepting stem were thermally stable under conditions of aminoacylation (Tms = 75 ± 1.5, 76 ± 0.9 and 83 ± 2.0 °C, respectively). In binding competition, the modified ASLGlu-s2U34 bound ERS with a higher affinity (half maximal inhibiting concentration, IC50, 5.1 ± 0.4 µM) than its unmodified counterpart, ASLGlu (IC50, 10.3 ± 0.6 µM). The minihelixGlu, ASLGlu-s2U34, and ASLGlu bound ERS with Kds of 9.9 ± 3.3, 6.5 ± 1.7 and 20.5 ± 3.8 µM. ERS aminoacylation of tRNAGlu was inhibited by the tRNA fragments. Unmodified ASLGlu, minihelixGlu, and ASLGlu-s2U34 exhibited a Kic of 1.9 ± 0.2 µM, 4.1 ± 0.2 µM, and 6.5 ± 0.1 µM, respectively. The modified ASLGlu-s2U34, though having a higher affinity for ERS, may be released more readily and thus, not be as good an inhibitor as the unmodified ASL. Thus, the RNA constructs are effective tools to study RNA-protein interaction.}, number={2}, journal={RNA BIOLOGY}, author={Gustilo, Estella M. and Dubois, Daniel Y. and Lapointe, Jacques and Agris, Paul F.}, year={2007}, pages={85–92} }