@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{harris_jones_bilbille_swairjo_agris_2011, title={YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase}, volume={17}, number={9}, journal={RNA}, author={Harris, K. A. and Jones, V. and Bilbille, Y. and Swairjo, M. A. and Agris, P. F.}, year={2011}, pages={1678–1687} }
 @article{scheunemann_graham_vendeix_agris_2010, title={Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA}, volume={38}, number={9}, journal={Nucleic Acids Research}, author={Scheunemann, A. E. and Graham, W. D. and Vendeix, F. A. P. and Agris, P. F.}, year={2010}, pages={3094–3105} }
 @article{vendeix_munoz_agris_2009, title={Free energy calculation of modified base-pair formation in explicit solvent: A predictive model}, volume={15}, ISSN={["1469-9001"]}, DOI={10.1261/rna.1734309}, abstractNote={The maturation of RNAs includes site-specific post-transcriptional modifications that contribute significantly to hydrogen bond formation within RNA and between different RNAs, especially in formation of mismatch base pairs. Thus, an understanding of the geometry and strength of the base-pairing of modified ribonucleoside 5′-monophosphates, previously not defined, is applicable to investigations of RNA structure and function and of the design of novel RNAs. The geometry and free energies of base-pairings were calculated in aqueous solution under neutral conditions with AMBER force fields and molecular dynamics simulations (MDSs). For example, unmodified uridines were observed to bind to uridine and cytidine with significant stability, but the ribose C1′–C1′ distances were far short (∼8.9 Å) of distances observed for canonical A-form RNA helices. In contrast, 5-oxyacetic acid uridine, known to bind adenosine, wobble to guanosine, and form mismatch base pairs with uridine and cytidine, bound adenosine and guanosine with geometries and energies comparable to an unmodified uridine. However, the 5-oxyacetic acid uridine base paired to uridine and cytidine with a C1′–C1′ distance comparable to that of an A-form helix, ∼11 Å, when a H2O molecule migrated between and stably hydrogen bonded to both bases. Even in formation of canonical base pairs, intermediate structures with a second energy minimum consisted of transient H2O molecules forming hydrogen bonded bridges between the two bases. Thus, MDS is predictive of the effects of modifications, H2O molecule intervention in the formation of base-pair geometry, and energies that are important for native RNA structure and function.}, number={12}, journal={RNA}, author={Vendeix, Franck A. P. and Munoz, Antonio M. and Agris, Paul F.}, year={2009}, month={Dec}, pages={2278–2287} }
 @article{bilbille_vendeix_guenther_malkiewicz_ariza_vilarrasa_agris_2009, title={The structure of the human tRNA(Lys3) anticodon bound to the HIV genome is stabilized by modified nucleosides and adjacent mismatch base pairs}, volume={37}, ISSN={["0305-1048"]}, DOI={10.1093/nar/gkp187}, abstractNote={Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNALys3, to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3′-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5′-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNALys3. The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s2U34, and pseudouridine, Ψ39, appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U162•Ψ39 and G163•A38, that maintained a reasonable A-form helix diameter. The tRNA's s2U34 stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Ψ39 stabilized the adjacent mismatched pairs.}, number={10}, journal={NUCLEIC ACIDS RESEARCH}, author={Bilbille, Yann and Vendeix, Franck A. P. and Guenther, Richard and Malkiewicz, Andrzej and Ariza, Xavier and Vilarrasa, Jaume and Agris, Paul F.}, year={2009}, month={Jun}, pages={3342–3353} }
 @article{jones_jones_graham_agris_spremulli_2008, title={A Disease-causing Point Mutation in Human Mitochondrial tRNA(Met) Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation}, volume={283}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M806992200}, abstractNote={The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNAMet (hmtRNAMet) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNAMet with a C8. The hmtRNAMet serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U→C mutation leads to a failure of the tRNA to respond conformationally to Mg2+. This mutation results in a drastic disruption of the structure of the hmtRNAMet, which significantly reduces its aminoacylation. The small fraction of hmtRNAMet that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg2+, the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg2+, the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg2+-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.}, number={49}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Jones, Christie N. and Jones, Christopher I. and Graham, William D. and Agris, Paul F. and Spremulli, Linda L.}, year={2008}, month={Dec}, pages={34445–34456} }
 @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{agris_2008, title={Bringing order to translation: the contributions of transfer RNA anticodon-domain modifications}, volume={9}, ISSN={["1469-3178"]}, DOI={10.1038/embor.2008.104}, abstractNote={The biosynthesis of RNA includes its post‐transcriptional modifications, and the crucial functions of these modifications have supported their conservation within all three kingdoms. For example, the modifications located within or adjacent to the anticodon of the transfer RNA (tRNA) enhance the accuracy of codon binding, maintain the translational reading frame and enable translocation of the tRNA from the A‐site to the P‐site of the ribosome. Although composed of different chemistries, the more than 70 known modifications of tRNA have in common their ability to reduce conformational dynamics, and to bring order to the internal loops and hairpin structures of RNA. The modified nucleosides of the anticodon domain of tRNA restrict its dynamics and shape its architecture; therefore, the need of the ribosome to constrain or remodel each tRNA to fit the decoding site is diminished. This concept reduces an entropic penalty for translation and provides a physicochemical basis for the conservation of RNA modifications in general.}, number={7}, journal={EMBO REPORTS}, author={Agris, Paul F.}, year={2008}, month={Jul}, pages={629–635} }
 @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} }
 @article{weixlbaumer_murphy_dziergowska_malkiewicz_vendeix_agris_ramakrishnan_2007, title={Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines}, volume={14}, ISSN={["1545-9985"]}, DOI={10.1038/nsmb1242}, abstractNote={One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.}, number={6}, journal={NATURE STRUCTURAL & MOLECULAR BIOLOGY}, author={Weixlbaumer, Albert and Murphy, Frank V. and Dziergowska, Agnieszka and Malkiewicz, Andrzej and Vendeix, Franck A. P. and Agris, Paul F. and Ramakrishnan, V.}, year={2007}, month={Jun}, pages={498–502} }
 @article{barley-maloney_agris_2007, title={Quality assessment of commercial small interfering RNA and DNA: Monoclonal antibodies and a high-throughput chemiluminescence assay}, volume={360}, number={1}, journal={Analytical Biochemistry}, author={Barley-Maloney, L. and Agris, P. F.}, year={2007}, pages={172–174} }
 @article{eshete_marchbank_deutscher_sproat_leszczynska_malkiewicz_agris_2007, title={Specificity of phage display selected peptides for modified anticodon stem and loop domains of tRNA}, volume={26}, ISSN={["1573-4943"]}, DOI={10.1007/s10930-006-9046-z}, abstractNote={Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA(LyS3) having 2-thiouridine (s(2)U34) and pseudouridine (psi39), bound the modified human ASL(Lys3)(s(2)U34;psi39) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL(LYS3)(s(2)U34; psi39), followed by the hypermodified ASL(Lys3) (mcm(5)s(2) U34; ms(2)t(6)A37) and the unmodified ASL(Lys3), but bound poorly to singly modified ASL(Lys3) constructs (psi39, ms(2)t(6)A37, s(2)34), ASL(Lys1,2) (t(6)A37) and Escherichia coli ASL(Glu) (s(2)U34). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.}, number={1}, journal={PROTEIN JOURNAL}, author={Eshete, Matthewos and Marchbank, Marie T. and Deutscher, Susan L. and Sproat, Brian and Leszczynska, Grazyna and Malkiewicz, Andrzej and Agris, Paul F.}, year={2007}, month={Jan}, pages={61–73} }
 @misc{agris_vendeix_graham_2007, title={tRNA's wobble decoding of the genome: 40 years of modification}, volume={366}, number={1}, journal={Journal of Molecular Biology}, author={Agris, P. F. and Vendeix, F. A. P. and Graham, W. D.}, year={2007}, pages={1–13} }
 @article{jones_spencer_hsu_spremulli_martinis_derider_agris_2006, title={A counterintuitive Mg2+-dependent and modification-assisted functional folding of mitochondrial tRNAs}, volume={362}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2006.07.036}, abstractNote={Mitochondrial tRNAs (mtRNAs) often lack domains and posttranscriptional modifications that are found in cytoplasmic tRNAs. These structural and chemical elements normally stabilize the folding of cytoplasmic tRNAs into canonical structures that are competent for aminoacylation and translation. For example, the dihydrouridine (D) stem and loop domain is involved in the tertiary structure of cytoplasmic tRNAs through hydrogen bonds and a Mg2+ bridge to the ribothymidine (T) stem and loop domain. These interactions are often absent in mtRNA because the D-domain is truncated or missing. Using gel mobility shift analyses, UV, circular dichroism and NMR spectroscopies and aminoacylation assays, we have investigated the functional folding interactions of chemically synthesized and site-specifically modified mitochondrial and cytoplasmic tRNAs. We found that Mg2+ is critical for folding of the truncated D-domain of bovine mtRNAMet with the tRNA's T-domain. Contrary to the expectation that Mg2+ stabilizes RNA folding, the mtRNAMet D-domain structure was unfolded and relaxed, rather than stabilized in the presence of Mg2+. Because the D-domain is transcribed prior to the T-domain, we conclude that Mg2+ prevents misfolding of the 5'-half of bovine mtRNAMet facilitating its correct interaction with the T-domain. The interaction of the mtRNAMet D-domain with the T-domain was enhanced by a pseudouridine located in either the D or T-domains compared to that of the unmodified RNAs (Kd=25.3, 24.6 and 44.4 microM, respectively). Mg2+ also affected the folding interaction of a yeast mtRNALeu1, but had minimal effect on the folding of an Escherichia coli cytoplasmic tRNALeu. The D-domain modification, dihydrouridine, facilitated mtRNALeu folding. These data indicate that conserved modifications assist and stabilize the formation of the functional mtRNA tertiary structure.}, number={4}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Jones, Christopher I. and Spencer, Angela C. and Hsu, Jennifer L. and Spremulli, Linda L. and Martinis, Susan A. and DeRider, Michele and Agris, Paul F.}, year={2006}, month={Sep}, pages={771–786} }
 @article{gagnon_zhang_agris_maxwell_2006, title={Assembly of the archaeal box C/D sRNP can occur via alternative pathways and requires temperature-facilitated sRNA remodeling}, volume={362}, DOI={10.1016/j.jmb.2006.07.091}, abstractNote={Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C'/D' motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or "remodeling" as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7-sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6's distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active complexes.}, number={5}, journal={Journal of Molecular Biology}, author={Gagnon, K. T. and Zhang, X. X. and Agris, P. F. and Maxwell, E. S.}, year={2006}, pages={1025–1042} }
 @article{dressman_barley-maloney_rowlette_agris_garcia-blanco_2006, title={Assessing incomplete deprotection of microarray oligonucleotides in situ}, volume={34}, number={19}, journal={Nucleic Acids Research}, author={Dressman, H. K. and Barley-Maloney, L. and Rowlette, L. L. and Agris, P. F. and Garcia-Blanco, M. A.}, year={2006} }
 @article{nelson_henkin_agris_2006, title={tRNA regulation of gene expression: Interactions of an mRNA 5 '-UTR with a regulatory tRNA}, volume={12}, ISSN={["1469-9001"]}, DOI={10.1261/rna.29906}, abstractNote={Many genes encoding aminoacyl-tRNA synthetases and other amino acid–related products in Gram-positive bacteria, including important pathogens, are regulated through interaction of unacylated tRNA with the 5′-untranslated region (5′-UTR) of the mRNA. Each gene regulated by this mechanism responds specifically to the cognate tRNA, and specificity is determined by pairing of the anticodon of the tRNA with a codon sequence in the “Specifier Loop” of the 5′-UTR. For the 5′-UTR to function in gene regulation, the mRNA folding interactions must be sufficiently stable to present the codon sequence for productive binding to the anticodon of the matching tRNA. A model bimolecular system was developed in which the interaction between two half molecules (“Common” and “Specifier”) would reconstitute the Specifier Loop region of the 5′-UTR of the Bacillus subtilis glyQS gene, encoding GlyRS mRNA. Gel mobility shift analysis and fluorescence spectroscopy yielded experimental Kds of 27.6 ± 1.0 μM and 10.5 ± 0.7 μM, respectively, for complex formation between Common and Specifier half molecules. The reconstituted 5′-UTR of the glyQS mRNA bound the anticodon stem and loop of tRNAGly (ASLGlyGCC) specifically and with a significant affinity (Kd = 20.2 ± 1.4 μM). Thus, the bimolecular 5′-UTR and ASLGlyGCC models mimic the RNA–RNA interaction required for T box gene regulation in vivo.}, number={7}, journal={RNA}, author={Nelson, Audrey R. and Henkin, Tina M. and Agris, Paul F.}, year={2006}, month={Jul}, pages={1254–1261} }
 @misc{agris_ashraf_2005, title={Antibacterial and antiviral agents and methods of screening for the same}, volume={6,962,785}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Agris, P. F. and Ashraf, S.}, year={2005} }
 @misc{agris_2005, title={Methods and compositions for determining the purity of chemically synthesized nucleic acids}, volume={6,929,907}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Agris, P. F.}, year={2005} }
 @misc{agris_2004, title={Decoding the genome: a modified view}, volume={32}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkh185}, abstractNote={Transfer RNA's role in decoding the genome is critical to the accuracy and efficiency of protein synthesis. Though modified nucleosides were identified in RNA 50 years ago, only recently has their importance to tRNA's ability to decode cognate and wobble codons become apparent. RNA modifications are ubiquitous. To date, some 100 different posttranslational modifications have been identified. Modifications of tRNA are the most extensively investigated; however, many other RNAs have modified nucleosides. The modifications that occur at the first, or wobble position, of tRNA's anticodon and those 3'-adjacent to the anticodon are of particular interest. The tRNAs most affected by individual and combinations of modifications respond to codons in mixed codon boxes where distinction of the third codon base is important for discriminating between the correct cognate or wobble codons and the incorrect near-cognate codons (e.g. AAA/G for lysine versus AAU/C asparagine). In contrast, other modifications expand wobble codon recognition, such as U*U base pairing, for tRNAs that respond to multiple codons of a 4-fold degenerate codon box (e.g. GUU/A/C/G for valine). Whether restricting codon recognition, expanding wobble, enabling translocation, or maintaining the messenger RNA, reading frame modifications appear to reduce anticodon loop dynamics to that accepted by the ribosome. Therefore, we suggest that anticodon stem and loop domain nucleoside modifications allow a limited number of tRNAs to accurately and efficiently decode the 61 amino acid codons by selectively restricting some anticodon-codon interactions and expanding others.}, number={1}, journal={NUCLEIC ACIDS RESEARCH}, author={Agris, PF}, year={2004}, month={Jan}, pages={223–238} }
 @article{phelps_malkiewicz_agris_joseph_2004, title={Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation}, volume={338}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2004.02.070}, abstractNote={Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.}, number={3}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Phelps, SS and Malkiewicz, A and Agris, PF and Joseph, S}, year={2004}, month={Apr}, pages={439–444} }
 @article{mucha_szyk_rekowski_agris_2004, title={Sequence-altered peptide adopts optimum conformation for modification-dependent binding of the yeast tRNA(Phe) anticodon domain}, volume={23}, ISSN={["1573-4943"]}, DOI={10.1023/B:JOPC.0000016256.20648.0f}, abstractNote={Amino acid contributions to protein recognition of naturally modified RNAs are not understood. Circular dichroism spectra and predictive software suggested that peptide tF2 (S1ISPW5GFSGL10 LRWSY15), selected from a phage display library to bind the modified anticodon domain of yeast tRNAPhe (ASL), adopted a beta-sheet structure. Ala residues incorporated at positions Pro4 and Gly6, both predicted to be involved in a turn, did not alter the peptide binding affinity for the ASLPhe, although major changes in the peptide's CD spectra were observed. Substitutions at three positions Pro4, Gly6, and Gly9, the latter not predicted to be in a turn, reduced the peptide's binding affinity to 4% of that of the unsubstituted tF2 and strongly influenced the peptide's secondary structure. The results suggest that peptides with different conformations, but similar affinities, adopt the optimal binding conformation, indicative of a structurally adaptive model of binding in which the modified RNA serves as a scaffold.}, number={1}, journal={PROTEIN JOURNAL}, author={Mucha, P and Szyk, A and Rekowski, P and Agris, PF}, year={2004}, month={Jan}, pages={33–38} }
 @article{guenther_sit_gracz_dolan_townsend_liu_newman_agris_lommel_2004, title={Structural characterization of an intermolecular RNA-RNA interaction involved in the transcription regulation element of a bipartite plant virus}, volume={32}, ISSN={["1362-4962"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-3042761419&partnerID=MN8TOARS}, DOI={10.1093/nar/gkh585}, abstractNote={The 34-nucleotide trans-activator (TA) located within the RNA-2 of Red clover necrotic mosaic virus folds into a simple hairpin. The eight-nucleotide TA loop base pairs with eight complementary nucleotides in the TA binding sequence (TABS) of the capsid protein subgenomic promoter on RNA-1 and trans-activates subgenomic RNA synthesis. Short synthetic oligoribonucleotide mimics of the RNA-1 TABS and the RNA-2 TA form a weak 1:1 bimolecular complex in vitro with a K(a) of 5.3 x 10(4) M(-1). K(a) determination for a series of RNA-1 and RNA-2 mimic variants indicated optimum stability is obtained with seven-base complementarity. Thermal denaturation and NMR show that the RNA-1 TABS 8mers are weakly ordered in solution while RNA-2 TA oligomers form the predicted hairpin. NMR diffusion studies confirmed RNA-1 and RNA-2 oligomer complex formation in vitro. MC-Sym generated structural models suggest that the bimolecular complex is composed of two stacked helices, one being the stem of the RNA-2 TA hairpin and the other formed by the intermolecular base pairing between RNA-1 and RNA-2. The RCNMV TA structural model is similar to those for the Simian retrovirus frameshifting element and the Human immunodeficiency virus-1 dimerization kissing hairpins, suggesting a conservation of form and function.}, number={9}, journal={NUCLEIC ACIDS RESEARCH}, publisher={Oxford University Press (OUP)}, author={Guenther, RH and Sit, TL and Gracz, HS and Dolan, MA and Townsend, HL and Liu, GH and Newman, WH and Agris, PF and Lommel, SA}, year={2004}, month={May}, pages={2819–2828} }
 @article{murphy_ramakrishnan_malkiewicz_agris_2004, title={The role of modifications in codon discrimination by tRNA(Lys) UUU}, volume={11}, ISSN={["1545-9985"]}, DOI={10.1038/nsmb861}, abstractNote={The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.}, number={12}, journal={NATURE STRUCTURAL & MOLECULAR BIOLOGY}, author={Murphy, FV and Ramakrishnan, V and Malkiewicz, A and Agris, PF}, year={2004}, month={Dec}, pages={1186–1191} }
 @article{stuart_koshlap_guenther_agris_2003, title={Naturally-occurring modification restricts the anticodon domain conformational space of tRNA(Phe)}, volume={334}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2003.09.058}, abstractNote={Post-transcriptional modifications contribute chemistry and structure to RNAs. Modifications of tRNA at nucleoside 37, 3'-adjacent to the anticodon, are particularly interesting because they facilitate codon recognition and negate translational frame-shifting. To assess if the functional contribution of a position 37-modified nucleoside defines a specific structure or restricts conformational flexibility, structures of the yeast tRNA(Phe) anticodon stem and loop (ASL(Phe)) with naturally occurring modified nucleosides differing only at position 37, ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)), and ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)), were determined by NMR spectroscopy and restrained molecular dynamics. The ASL structures had similarly resolved stems (RMSD approximately 0.6A) of five canonical base-pairs in standard A-form RNA. The "NOE walk" was evident on the 5' and 3' sides of the stems of both RNAs, and extended to the adjacent loop nucleosides. The NOESY cross-peaks involving U(33) H2' and characteristic of tRNA's anticodon domain U-turn were present but weak, whereas those involving the U(33) H1' proton were absent from the spectra of both ASLs. However, ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) exhibited the downfield shifted 31P resonance of U(33)pGm(34) indicative of U-turns; ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)) did not. An unusual "backwards" NOE between Gm(34) and A(35) (Gm(34)/H8 to A(35)/H1') was observed in both molecules. The RNAs exhibited a protonated A(+)(38) resulting in the final structures having C(32).A(+)(38) intra-loop base-pairs, with that of ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) being especially well defined. A single family of low-energy structures of ASL(Phe)-(Cm(32),Gm(34), m(1)G(37),m(5)C(40)) (loop RMSD 0.98A) exhibited a significantly restricted conformational space for the anticodon loop in comparison to that of ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)) (loop RMSD 2.58A). In addition, the ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) average structure had a greater degree of similarity to that of the yeast tRNA(Phe) crystal structure. A comparison of the resulting structures indicates that modification of position 37 affects the accuracy of decoding and the maintenance of the mRNA reading frame by restricting anticodon loop conformational space.}, number={5}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Stuart, JW and Koshlap, KM and Guenther, R and Agris, PF}, year={2003}, month={Dec}, pages={901–918} }
 @article{mucha_szyk_rekowski_agris_2003, title={Using capillary electrophoresis to study methylation effect on RNA-peptide interaction}, volume={50}, number={3}, journal={Acta Biochimica Polonica}, author={Mucha, P. and Szyk, A. and Rekowski, P. and Agris, P. F.}, year={2003}, pages={857–864} }
 @article{yarian_townsend_czestkowski_sochacka_malkiewicz_guenther_miskiewicz_agris_2002, title={Accurate translation of the genetic code depends on tRNA modified nucleosides}, volume={277}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.M200253200}, abstractNote={Transfer RNA molecules translate the genetic code by recognizing cognate mRNA codons during protein synthesis. The anticodon wobble at position 34 and the nucleotide immediately 3′ to the anticodon triplet at position 37 display a large diversity of modified nucleosides in the tRNAs of all organisms. We show that tRNA species translating 2-fold degenerate codons require a modified U34 to enable recognition of their cognate codons ending in A or G but restrict reading of noncognate or near-cognate codons ending in U and C that specify a different amino acid. In particular, the nucleoside modifications 2-thiouridine at position 34 (s2U34), 5-methylaminomethyluridine at position 34 (mnm5U34), and 6-threonylcarbamoyladenosine at position 37 (t6A37) were essential for Watson-Crick (AAA) and wobble (AAG) cognate codon recognition by tRNA UUU Lys at the ribosomal aminoacyl and peptidyl sites but did not enable the recognition of the asparagine codons (AAU and AAC). We conclude that modified nucleosides evolved to modulate an anticodon domain structure necessary for many tRNA species to accurately translate the genetic code.}, number={19}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Yarian, C and Townsend, H and Czestkowski, W and Sochacka, E and Malkiewicz, AJ and Guenther, R and Miskiewicz, A and Agris, PF}, year={2002}, month={May}, pages={16391–16395} }
 @article{nobles_yarian_liu_guenther_agris_2002, title={Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding}, volume={30}, ISSN={["0305-1048"]}, DOI={10.1093/nar/gkf595}, abstractNote={Transfer RNA structure involves complex folding interactions of the TPsiC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TPsiC domain, rT54, Psi55 and m5C49, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA(Phe) T-half molecules (nucleosides 40-72) with the corresponding unmodified D-half molecule (nucleosides 1-30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg2+ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K(d)s (rT54 = 6 +/- 2, m5C49 = 11 +/- 2, Psi55 = 14 +/- 5, and rT54,Psi55 = 11 +/- 3 microM) significantly lower than that of the unmodified T-half (40 +/- 10 microM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA(Phe) and native yeast tRNA(Phe), as determined by lead cleavage patterns at U17 and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA's TPsiC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.}, number={21}, journal={NUCLEIC ACIDS RESEARCH}, author={Nobles, KN and Yarian, CS and Liu, G and Guenther, RH and Agris, PF}, year={2002}, month={Nov}, pages={4751–4760} }
 @article{fu_smith_simkins_agris_2002, title={Identification and quantification of protecting groups remaining in commercial oligonucleotide products using monoclonal antibodies}, volume={306}, ISSN={["0003-2697"]}, DOI={10.1006/abio.2002.5687}, abstractNote={Quality control is paramount to reproducibly achieving oligonucleotide therapeutics and diagnostics of superior value. However, incomplete deprotection of nucleoside reactive groups after the automated chemical synthesis of oligonucleotides would result in diminished antisense activity and in erroneous array analysis of gene expression. Mass spectrometry and capillary electrophoresis are used to detect aborted sequences of oligonucleotides, but not to identify and quantify incompletely deprotected oligonucleotides. To address this problem, monoclonal antibodies (MAbs), ELISA, and dot-blot assays were developed for the specific identification and quantification of the commonly used nucleic acid base- and sugar-protecting groups: benzoyl, isobutyryl, isopropylphenoxyacetyl, and dimethoxytrityl. Each MAb was capable of reproducibly detecting 8-32 pmol of the respectively protected nucleoside in an intact DNA or RNA sample composed of 320-640 nmol of the deprotected nucleoside. In a direct comparison, HPLC nucleoside composition analysis of enzyme-hydrolyzed DNA was limited to the detection of 2-5 nmol of protected nucleoside. Using the MAb dot-blot assay, 5 of 16 commercial DNA products obtained from eight different companies were found to have 1.0-5.2% of the benzoyl and isopropylphenoxyacetyl protecting groups remaining. Thus, MAbs selectively identify and quantify picomoles of remaining protecting groups on antisense therapeutics and oligonucleotide diagnostics.}, number={1}, journal={ANALYTICAL BIOCHEMISTRY}, author={Fu, C and Smith, S and Simkins, SG and Agris, PF}, year={2002}, month={Jul}, pages={135–143} }
 @article{mucha_szyk_rekowski_guenther_agris_2002, title={Interaction of RNA with phage display selected peptides analyzed by capillary electrophoresis mobility shift assay}, volume={8}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838202020319}, abstractNote={A sensitive capillary electrophoresis mobility shift assay (CEMSA) to analyze RNA/peptide interactions has been developed. Capillary electrophoresis (CE) has been adapted for investigating the interaction between variously methylated 17-nt analogs of the yeast tRNAPhe anticodon stem and loop domain (ASL(Phe)) and 15-amino-acid peptides selected from a random phage display library (RPL). A peptide-concentration-dependent formation of RNA/peptide complex was clearly visible during CEMSA. In the presence of peptide, the UV-monitored CE peak for ASLPhe with three of the five naturally occurring modifications (2'-O-methylcytidine (Cm32), 2'-O-methylguanine (Gm34) and 5-methylcytidine (m5C40) shifted from 18.16 to 20.90 min. The mobility shift was observed only for methylated RNA. The negative effects of diffusion, electroosmotic flow and adhesion of molecules to the capillary internal wall were suppressed by using a buffer containing a sieving polymer and a polyacrylamide-coated capillary. Under these conditions, well-shaped peaks and resolution of RNA free and bound to peptide were achieved. Peptide tF2, the most populated ligand in the RPL, specifically bound triply methylated ASLPhe in a methylated nucleoside-dependent manner. CE was found to be an efficient and sensitive method for the qualitative analysis of RNA-peptide interaction and should be generally applicable to the study of RNA-peptide (protein) interactions.}, number={5}, journal={RNA}, author={Mucha, P and Szyk, A and Rekowski, P and Guenther, R and Agris, PF}, year={2002}, month={May}, pages={698–704} }
 @misc{agris_smith_fu_simkins_2002, title={QC in antisense oligo synthesis}, volume={20}, number={9}, journal={Nature Biotechnology}, author={Agris, P. F. and Smith, S. and Fu, C. and Simkins, S. G.}, year={2002}, pages={871–872} }
 @article{mucha_szyk_rekowski_weiss_agris_2001, title={Anticodon domain methylated nucleosides of yeast tRNA(Phe) are significant recognition determinants in the binding of a phage display selected peptide}, volume={40}, ISSN={["0006-2960"]}, DOI={10.1021/bi010978o}, abstractNote={The contributions of the natural modified nucleosides to RNA identity in protein/RNA interactions are not understood. We had demonstrated that 15 amino acid long peptides could be selected from a random phage display library using the criterion of binding to a modified, rather than unmodified, anticodon domain of yeast tRNA(Phe) (ASL(Phe)). Affinity and specificity of the selected peptides for the modified ASL(Phe) have been characterized by fluorescence spectroscopy of the peptides' tryptophans. One of the peptides selected, peptide t(F)2, exhibited the highest specificity and most significant affinity for ASL(Phe) modified with 2'-O-methylated cytidine-32 and guanosine-34 (Cm(32) and Gm(34)) and 5-methylated cytidine-40 (m(5)C(40)) (K(d) = 1.3 +/- 0.4 microM) and a doubly modified ASL(Phe)-Gm(34),m(5)C(40) and native yeast tRNA(Phe) (K(d) congruent with 2.3 and 3.8 microM, respectively) in comparison to that for the unmodified ASL(Phe) (K(d) = 70.1 +/- 12.3 microM). Affinity was reduced when a modification altered the ASL loop structure, and binding was negated by modifications that disfavored hairpin formation. Peptide t(F)2's higher affinity for the ASL(Phe)-Cm(32),Gm(34),m(5)C(40) hairpin and fluorescence resonance energy transfer from its tryptophan to the hypermodified wybutosine-37 in the native tRNA(Phe) placed the peptide across the anticodon loop and onto the 3'-side of the stem. Inhibition of purified yeast phenylalanyl-tRNA synthetase (FRS) catalyzed aminoacylation of cognate yeast tRNA(Phe) corroborated the peptide's binding to the anticodon domain. The phage-selected peptide t(F)2 has three of the four amino acids crucial to G(34) recognition by the beta-structure of the anticodon-binding domain of Thermus thermophilus FRS and exhibited circular dichroism spectral properties characteristic of beta-structure. Thus, modifications as simple as methylations contribute identity elements that a selected peptide specifically recognizes in binding synthetic and native tRNA and in inhibiting tRNA aminoacylation.}, number={47}, journal={BIOCHEMISTRY}, author={Mucha, P and Szyk, A and Rekowski, P and Weiss, PA and Agris, PF}, year={2001}, month={Nov}, pages={14191–14199} }
 @article{stuart_gdaniec_guenther_marszalek_sochacka_malkiewicz_agris_2000, title={Functional anticodon architecture of human tRNA(Lys3) includes disruption of intraloop hydrogen bonding by the naturally occurring amino acid modification, t(6)A}, volume={39}, ISSN={["0006-2960"]}, DOI={10.1021/bi0013039}, abstractNote={The structure of the human tRNA(Lys3) anticodon stem and loop domain (ASL(Lys3)) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t(6)A(37)) to tRNA(Lys3) functions. The t(6)A(37)-modified anticodon stem and loop domain of tRNA(Lys3)(UUU) (ASL(Lys3)(UUU)- t(6)A(37)) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASL(Lys3)(UUU) is not [Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390-13395]. The structure, determined to an average rmsd of 1.57 +/- 0.33 A (relative to the mean structure) by NMR spectroscopy and restrained molecular dynamics, is the first reported of an RNA in which a naturally occurring hypermodified nucleoside was introduced by automated chemical synthesis. The ASL(Lys3)(UUU)-t(6)A(37) loop is significantly different than that of the unmodified ASL(Lys3)(UUU), although the five canonical base pairs of both ASL(Lys3)(UUU) stems are in the standard A-form of helical RNA. t(6)A(37), 3'-adjacent to the anticodon, adopts the form of a tricyclic nucleoside with an intraresidue H-bond and enhances base stacking on the 3'-side of the anticodon loop. Critically important to ribosome binding, incorporation of the modification negates formation of an intraloop U(33).A(37) base pair that is observed in the unmodified ASL(Lys3)(UUU). The anticodon wobble position U(34) nucleobase in ASL(Lys3)(UUU)-t(6)A(37) is significantly displaced from its position in the unmodified ASL and directed away from the codon-binding face of the loop resulting in only two anticodon bases for codon binding. This conformation is one explanation for ASL(Lys3)(UUU) tendency to prematurely terminate translation and -1 frame shift. At the pH 5.6 conditions of our structure determination, A(38) is protonated and positively charged in ASL(Lys3)(UUU)-t(6)A(37) and the unmodified ASL(Lys3)(UUU). The ionized carboxylic acid moiety of t(6)A(37) possibly neutralizes the positive charge of A(+)(38). The protonated A(+)(38) can base pair with C(32), but t(6)A(37) may weaken the interaction through steric interference. From these results, we conclude that ribosome binding cannot simply be an induced fit of the anticodon stem and loop, otherwise the unmodified ASL(Lys3)(UUU) would bind as well as ASL(Lys3)(UUU)-t(6)A(37). t(6)A(37) and other position 37 modifications produce the open, structured loop required for ribosomal binding.}, number={44}, journal={BIOCHEMISTRY}, author={Stuart, JW and Gdaniec, Z and Guenther, R and Marszalek, M and Sochacka, E and Malkiewicz, A and Agris, PF}, year={2000}, month={Nov}, pages={13396–13404} }
 @article{sengupta_vainauskas_yarian_sochacka_malkiewicz_guenther_koshlap_agris_2000, title={Modified constructs of the tRNA T Psi C domain to probe substrate conformational requirements of m(1)A(58) and m(5)U(54) tRNA methyltransferases}, volume={28}, ISSN={["0305-1048"]}, DOI={10.1093/nar/28.6.1374}, abstractNote={The TPsiC stem and loop (TSL) of tRNA contains highly conserved nucleoside modifications, m(5)C(49), T(54), Psi(55)and m(1)A(58). U(54)is methylated to m(5)U (T) by m(5)U(54)methyltransferase (RUMT); A(58)is methylated to m(1)A by m(1)A(58)tRNA methyltransferase (RAMT). RUMT recognizes and methylates a minimal TSL heptadecamer and RAMT has previously been reported to recognize and methylate the 3'-half of the tRNA molecule. We report that RAMT can recognize and methylate a TSL heptadecamer. To better understand the sensitivity of RAMT and RUMT to TSL conformation, we have designed and synthesized variously modified TSL constructs with altered local conformations and stabilities. TSLs were synthesized with natural modifications (T(54)and Psi(55)), naturally occurring modifications at unnatural positions (m(5)C(60)), altered sugar puckers (dU(54)and/or dU(55)) or with disrupted U-turn interactions (m(1)Psi(55)or m(1)m(3)Psi(55)). The unmodified heptadecamer TSL was a substrate of both RAMT and RUMT. The presence of T(54)increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate. Local conformation around U(54)was found to be an important determinant for the activities of both RAMT and RUMT.}, number={6}, journal={NUCLEIC ACIDS RESEARCH}, author={Sengupta, R and Vainauskas, S and Yarian, C and Sochacka, E and Malkiewicz, A and Guenther, RH and Koshlap, KM and Agris, PF}, year={2000}, month={Mar}, pages={1374–1380} }
 @article{yarian_marszalek_sochacka_malkiewicz_guenther_miskiewicz_agris_2000, title={Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNA(UUU)(Lys) species}, volume={39}, ISSN={["0006-2960"]}, DOI={10.1021/bi001302g}, abstractNote={Nucleoside modifications are important to the structure of all tRNAs and are critical to the function of some tRNA species. The transcript of human tRNA(Lys3)(UUU) with a UUU anticodon, and the corresponding anticodon stem and loop domain (ASL(Lys3)(UUU)), are unable to bind to poly-A programmed ribosomes. To determine if specific anticodon domain modified nucleosides of tRNA(Lys) species would restore ribosomal binding and also affect thermal stability, we chemically synthesized ASL(Lys) heptadecamers and site-specifically incorporated the anticodon domain modified nucleosides pseudouridine (Psi(39)), 5-methylaminomethyluridine (mnm(5)U(34)) and N6-threonylcarbamoyl-adenosine (t(6)A(37)). Incorporation of t(6)A(37) and mnm(5)U(34) contributed structure to the anticodon loop, apparent by increases in DeltaS, and significantly enhanced the ability of ASL(Lys3)(UUU) to bind poly-A programmed ribosomes. Neither ASL(Lys3)(UUU)-t(6)A(37) nor ASL(Lys3)(UUU)-mnm(5)U(34) bound AAG programmed ribosomes. Only the presence of both t(6)A(37) and mnm(5)U(34) enabled ASL(Lys3)(UUU) to bind AAG programmed ribosomes, as well as increased its affinity for poly-A programmed ribosomes to the level of native Escherichia coli tRNA(Lys). The completely unmodified anticodon stem and loop of human tRNA(Lys1,2)(CUU) with a wobble position-34 C bound AAG, but did not wobble to AAA, even when the ASL was modified with t(6)A(37). The data suggest that tRNA(Lys)(UUU) species require anticodon domain modifications in the loop to impart an ordered structure to the anticodon for ribosomal binding to AAA and require a combination of modified nucleosides to bind AAG.}, number={44}, journal={BIOCHEMISTRY}, author={Yarian, C and Marszalek, M and Sochacka, E and Malkiewicz, A and Guenther, R and Miskiewicz, A and Agris, PF}, year={2000}, month={Nov}, pages={13390–13395} }
 @article{ashraf_guenther_ansari_malkiewicz_sochacka_agris_2000, title={Role of modified nucleosides of yeast tRNA(Phe) in ribosomal binding}, volume={33}, ISSN={["1559-0283"]}, DOI={10.1385/CBB:33:3:241}, abstractNote={Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.}, number={3}, journal={CELL BIOCHEMISTRY AND BIOPHYSICS}, author={Ashraf, SS and Guenther, RH and Ansari, G and Malkiewicz, A and Sochacka, E and Agris, PF}, year={2000}, pages={241–252} }
 @article{sochacka_czerwinska_guenther_cain_agris_malkiewicz_2000, title={Synthesis and properties of uniquely modified oligoribonucleotides: Yeast tRNA(Phe) fragments with 6-methyluridine and 5,6-dimethyluridine at site-specific positions}, volume={19}, ISSN={["1525-7770"]}, DOI={10.1080/15257770008035004}, abstractNote={Abstract The phosphoramidites of 6-methyluridine and 5,6-dimethyluridine were synthesized and the modified uridines site-selectively incorporated into heptadecamers corresponding in sequence to the yeast tRNAPhe anticodon and TΦC domains. The oligoribonucleotides were characterized by NMR, MALDI-TOF MS and UV-monitored thermal denaturations. The 6-methylated uridines retained the syn conformation at the polymer level and in each sequence location destabilized the RNAs compared to that of the unmodified RNA. The decrease in RNA duplex stability is predictable. However, loss of stability when the modified uridine is in a loop is sequence context dependent, and can not, at this time, be predicted from the location in the loop.}, number={3}, journal={NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS}, author={Sochacka, E and Czerwinska, G and Guenther, R and Cain, R and Agris, PF and Malkiewicz, A}, year={2000}, pages={515–531} }
 @article{koshlap_guenther_sochacka_malkiewicz_agris_1999, title={A distinctive RNA fold: The solution structure of an analogue of the yeast tRNA(Phe) T psi C domain}, volume={38}, ISSN={["0006-2960"]}, DOI={10.1021/bi990118w}, abstractNote={The structure of an analogue of the yeast tRNAPhe T Psi C stem-loop has been determined by NMR spectroscopy and restrained molecular dynamics. The molecule contained the highly conserved modification ribothymidine at its naturally occurring position. The ribothymidine-modified T Psi C stem-loop is the product of the m5U54-tRNA methyltransferase, but is not a substrate for the m1A58-tRNA methyltransferase. Site-specific substitutions and 15N labels were used to confirm the assignment of NOESY cross-peaks critical in defining the global fold of the molecule. The structure is unusual in that the loop folds far over into the major groove of the curved stem. This conformation is stabilized by both stacking interactions and hydrogen bond formation. Furthermore, this conformation appears to be unique among RNA hairpins of similar size. There is, however, a considerable resemblance to the analogous domain in the crystal structure of the full-length yeast tRNAPhe. We believe, therefore, that the structure we have determined may represent an intermediate in the folding pathway during the maturation of tRNA.}, number={27}, journal={BIOCHEMISTRY}, author={Koshlap, KM and Guenther, R and Sochacka, E and Malkiewicz, A and Agris, PF}, year={1999}, month={Jul}, pages={8647–8656} }
 @misc{agris_ashraf_1999, title={Antibacterial agents and methods of screening for the same}, volume={6,461,815}, number={1999 May 20}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Agris, P. F. and Ashraf, S.}, year={1999} }
 @article{agris_marchbank_newman_guenther_ingram_swallow_mucha_szyk_rekowski_peletskaya_et al._1999, title={Experimental models of protein-RNA interaction: Isolation and analyses of tRNA(Phe) and U1 snRNA-binding peptides from bacteriophage display libraries}, volume={18}, ISSN={["0277-8033"]}, DOI={10.1023/A:1020688609121}, abstractNote={Peptides that bind either U1 small nuclear RNA (U1 snRNA) or the anticodon stem and loop of yeast tRNA(Phe) (tRNA(ACPhe)) were selected from a random-sequence, 15-amino acid bacteriophage display library. An experimental system, including an affinity selection method, was designed to identify primary RNA-binding peptide sequences without bias to known amino acid sequences and without incorporating nonspecific binding of the anionic RNA backbone. Nitrocellulose binding assays were used to evaluate the binding of RNA by peptide-displaying bacteriophage. Amino acid sequences of RNA-binding bacteriophage were determined from the foreign insert DNA sequences, and peptides corresponding to the RNA-binding bacteriophage inserts were chemically synthesized. Peptide affinities for the RNAs (Kd approximately 0.1-5.0 microM) were analyzed successfully using fluorescence and circular dichroism spectroscopies. These methodologies demonstrate the feasibility of rapidly identifying, isolating, and initiating the analyses of small peptides that bind to RNAs in an effort to define better the chemistry, structure, and function of protein-RNA complexes.}, number={4}, journal={JOURNAL OF PROTEIN CHEMISTRY}, author={Agris, PF and Marchbank, MT and Newman, W and Guenther, R and Ingram, P and Swallow, J and Mucha, P and Szyk, A and Rekowski, P and Peletskaya, E and et al.}, year={1999}, month={May}, pages={425–435} }
 @article{ashraf_guenther_agris_1999, title={Orientation of the tRNA anticodon in the ribosomal P-site: Quantitative footprinting with U-33-modified, anticodon stem and loop domains}, volume={5}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838299990933}, abstractNote={Binding of transfer RNA (tRNA) to the ribosome involves crucial tRNA-ribosomal RNA (rRNA) interactions. To better understand these interactions, U33-substituted yeast tRNA(Phe) anticodon stem and loop domains (ASLs) were used as probes of anticodon orientation on the ribosome. Orientation of the anticodon in the ribosomal P-site was assessed with a quantitative chemical footprinting method in which protection constants (Kp) quantify protection afforded to individual 16S rRNA P-site nucleosides by tRNA or synthetic ASLs. Chemical footprints of native yeast tRNA(Phe), ASL-U33, as well as ASLs containing 3-methyluridine, cytidine, or deoxyuridine at position 33 (ASL-m3U33, ASL-C33, and ASL-dU33, respectively) were compared. Yeast tRNAPhe and the ASL-U33 protected individual 16S rRNA P-site nucleosides differentially. Ribosomal binding of yeast tRNA(Phe) enhanced protection of C1400, but the ASL-U33 and U33-substituted ASLs did not. Two residues, G926 and G1338 with KpS approximately 50-60 nM, were afforded significantly greater protection by both yeast tRNA(Phe) and the ASL-U33 than other residues, such as A532, A794, C795, and A1339 (KpS approximately 100-200 nM). In contrast, protections of G926 and G1338 were greatly and differentially reduced in quantitative footprints of U33-substituted ASLs as compared with that of the ASL-U33. ASL-m3U33 and ASL-C33 protected G530, A532, A794, C795, and A1339 as well as the ASL-U33. However, protection of G926 and G1338 (KpS between 70 and 340 nM) was significantly reduced in comparison to that of the ASL-U33 (43 and 61 nM, respectively). Though protections of all P-site nucleosides by ASL-dU33 were reduced as compared to that of the ASL-U33, a proportionally greater reduction of G926 and G1338 protections was observed (KpS = 242 and 347 nM, respectively). Thus, G926 and G1338 are important to efficient P-site binding of tRNA. More importantly, when tRNA is bound in the ribosomal P-site, G926 and G1338 of 16S rRNA and the invariant U33 of tRNA are positioned close to each other.}, number={9}, journal={RNA}, author={Ashraf, SS and Guenther, R and Agris, PF}, year={1999}, month={Sep}, pages={1191–1199} }
 @article{ashraf_sochacka_cain_guenther_malkiewicz_agris_1999, title={Single atom modification (O -> S) of tRNA confers ribosome binding}, volume={5}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838299981529}, abstractNote={Escherichia coli tRNALysSUU, as well as human tRNALys3SUU, has 2-thiouridine derivatives at wobble position 34 (s2U*34). Unlike the native tRNALysSUU, the full-length, unmodified transcript of human tRNALys3UUU and the unmodified tRNALys3UUU anticodon stem/loop (ASLLys3UUU) did not bind AAA- or AAG-programmed ribosomes. In contrast, the completely unmodified yeast tRNAPhe anticodon stem/loop (ASLPheGAA) had an affinity (Kd = 136+/-49 nM) similar to that of native yeast tRNAPheGmAA (Kd = 103+/-19 nM). We have found that the single, site-specific substitution of s2U34 for U34 to produce the modified ASLLysSUU was sufficient to restore ribosomal binding. The modified ASLLysSUU bound the ribosome with an affinity (Kd = 176+/-62 nM) comparable to that of native tRNALysSUU (Kd = 70+/-7 nM). Furthermore, in binding to the ribosome, the modified ASLLys3SUU produced the same 16S P-site tRNA footprint as did native E. coli tRNALysSUU, yeast tRNAPheGmAA, and the unmodified ASLPheGAA. The unmodified ASLLys3UUU had no footprint at all. Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASLLys3SUU was different from that of the unmodified ASLLysUUU, whereas the stems were isomorphous. Based on these and other data, we conclude that s2U34 in tRNALysSUU and in other s2U34-containing tRNAs is critical for generating an anticodon conformation that leads to effective codon interaction in all organisms. This is the first example of a single atom substitution (U34-->s2U34) that confers the property of ribosomal binding on an otherwise inactive tRNA.}, number={2}, journal={RNA}, author={Ashraf, SS and Sochacka, E and Cain, R and Guenther, R and Malkiewicz, A and Agris, PF}, year={1999}, month={Feb}, pages={188–194} }
 @article{yarian_basti_cain_ansari_guenther_sochacka_czerwinska_malkiewicz_agris_1999, title={Structural and functional roles of the N1-and N3-protons of Psi at tRNA's position 39}, volume={27}, ISSN={["1362-4962"]}, DOI={10.1093/nar/27.17.3543}, abstractNote={Pseudouridine at position 39 (Psi(39)) of tRNA's anticodon stem and loop domain (ASL) is highly conserved. To determine the physicochemical contributions of Psi(39)to the ASL and to relate these properties to tRNA function in translation, we synthesized the unmodified yeast tRNA(Phe)ASL and ASLs with various derivatives of U(39)and Psi(39). Psi(39)increased the thermal stability of the ASL (Delta T (m)= 1.3 +/- 0.5 degrees C), but did not significantly affect ribosomal binding ( K (d)= 229 +/- 29 nM) compared to that of the unmodified ASL (K (d)= 197 +/- 58 nM). The ASL-Psi(39)P-site fingerprint on the 30S ribosomal subunit was similar to that of the unmodified ASL. The stability, ribosome binding and fingerprint of the ASL with m(1)Psi(39)were comparable to that of the ASL with Psi(39). Thus, the contribution of Psi(39)to ASL stability is not related to N1-H hydrogen bonding, but probably is due to the nucleoside's ability to improve base stacking compared to U. In contrast, substitutions of m(3)Psi(39), the isosteric m(3)U(39)and m(1)m(3)Psi(39)destabilized the ASL by disrupting the A(31)-U(39)base pair in the stem, as confirmed by NMR. N3-methylations of both U and Psi dramatically decreased ribosomal binding ( K (d)= 1060 +/- 189 to 1283 +/- 258 nM). Thus, canonical base pairing of Psi(39)to A(31)through N3-H is important to structure, stability and ribosome binding, whereas the increased stability and the N1-proton afforded by modification of U(39)to Psi(39)may have biological roles other than tRNA's binding to the ribosomal P-site.}, number={17}, journal={NUCLEIC ACIDS RESEARCH}, author={Yarian, CS and Basti, MM and Cain, RJ and Ansari, G and Guenther, RH and Sochacka, E and Czerwinska, G and Malkiewicz, A and Agris, PF}, year={1999}, month={Sep}, pages={3543–3549} }
 @article{ashraf_ansari_guenther_sochacka_malkiewicz_agris_1999, title={The uridine in "U-turn": Contributions to tRNA-ribosomal binding}, volume={5}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838299981931}, abstractNote={"U-turns" represent an important class of structural motifs in the RNA world, wherein a uridine is involved in an abrupt change in the direction of the polynucleotide backbone. In the crystal structure of yeast tRNAPhe, the invariant uridine at position 33 (U33), adjacent to the anticodon, stabilizes the exemplar U-turn with three non-Watson-Crick interactions: hydrogen bonding of the 2'-OH to N7 of A35 and the N3-H to A36-phosphate, and stacking between C32 and A35-phosphate. The functional importance of each noncanonical interaction was determined by assaying the ribosomal binding affinities of tRNAPhe anticodon stem and loop domains (ASLs) with substitutions at U33. An unsubstituted ASL bound 30S ribosomal subunits with an affinity (Kd = 140+/-50 nM) comparable to that of native yeast tRNAPhe (Kd = 100+/-20 nM). However, the binding affinities of ASLs with dU-33 (no 2'-OH) and C-33 (no N3-H) were significantly reduced (2,930+/-140 nM and 2,190+/-300 nM, respectively). Surprisingly, the ASL with N3-methyluridine-33 (no N3-H) bound ribosomes with a high affinity (Kd = 220+/-20 nM). In contrast, ASLs constructed with position 33 uridine analogs in nonstacking, nonnative, and constrained conformations, dihydrouridine (C2'-endo), 6-methyluridine (syn) and 2'O-methyluridine (C3'-endo) had almost undetectable binding. The inability of ASLs with 6-methyluridine-33 and 2'O-methyluridine-33 to bind ribosomes was not attributable to any thermal instability of the RNAs. These results demonstrate that proton donations by the N3-H and 2'OH groups of U33 are not absolutely required for ribosomal binding. Rather, the results suggest that the overall uridine conformation, including a dynamic (C3'-endo > C2'-endo) sugar pucker, anti conformation, and ability of uracil to stack between C32 and A35-phosphate, are the contributing factors to a functional U-turn.}, number={4}, journal={RNA}, author={Ashraf, SS and Ansari, G and Guenther, R and Sochacka, E and Malkiewicz, A and Agris, PF}, year={1999}, month={Apr}, pages={503–511} }
 @article{agris_guenther_sochacka_newman_czerwinska_liu_ye_malkiewicz_1999, title={Thermodynamic contribution of nucleoside modifications to yeast tRNA(Phe) anticodon stem loop analogs}, volume={46}, number={1}, journal={Acta Biochimica Polonica}, author={Agris, P. F. and Guenther, R. and Sochacka, E. and Newman, W. and Czerwinska, G. and Liu, G. H. and Ye, W. P. and Malkiewicz, A.}, year={1999}, pages={163–172} }
 @article{guenther_forrest_newman_malkiewicz_agris_1998, title={Modified RNAs as potential drug targets}, volume={45}, number={1}, journal={Acta Biochimica Polonica}, author={Guenther, R. and Forrest, B. and Newman, W. and Malkiewicz, A. and Agris, P. F.}, year={1998}, pages={13–18} }
 @inproceedings{ashraf_guenther_ye_lee_malkiewicz_agris_1997, title={Ribosomal binding of modified tRNA anticodons related to thermal stability}, volume={36}, booktitle={Symposium on RNA Biology II. RNA: Tool and Target (1997: North Carolina Biotechnology Center) Research Triangle Park, North Carolina, USA, October 17-19, 1997  (Nucleic acids symposium series; no. 36)}, publisher={Oxford: Oxford University Press}, author={Ashraf, S. S. and Guenther, R. and Ye, W. and Lee, Y. and Malkiewicz, A. and Agris, P. F.}, year={1997}, pages={58–60} }
 @article{agris_guenther_ingram_basti_stuart_sochacka_malkiewicz_1997, title={Unconventional structure of tRNA(Lys)SUU anticodon explains tRNA's role in bacterial and mammalian ribosomal frameshifting and primer selection by HIV-1}, volume={3}, number={4}, journal={RNA}, author={Agris, P. F. and Guenther, R. H. and Ingram, P. C. and Basti, M. M. and Stuart, J. W. and Sochacka, E. and Malkiewicz, A.}, year={1997}, pages={420–428} }