@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 ( approximately 8.9 A) 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, approximately 11 A, when a H(2)O 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 H(2)O molecules forming hydrogen bonded bridges between the two bases. Thus, MDS is predictive of the effects of modifications, H(2)O 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. 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. Human mitochondria are subcellular organelles that produce more than 90% of the energy required by the cell. The mitochondrial genome encodes 13 proteins necessary for energy production, two rRNAs and all of the 22 tRNAs required for the synthesis of these proteins (1Attardi G. Int. Rev. Cytol. 1985; 93: 93-145Crossref PubMed Scopus (237) Google Scholar, 2Anderson S. de Brujin M. Coulson A. Eperon I. Sanger F. Young I. J. Mol. Biol. 1982; 156: 683-717Crossref PubMed Scopus (1188) Google Scholar). Mammalian mitochondrial tRNAs have several unusual features that distinguish them from canonical tRNAs. In many cases, they lack a number of the conserved or semi-conserved nucleotides that play important roles in creating the L-shaped tertiary structure of prokaryotic and eukaryotic cytoplasmic tRNAs (3Dirheimer G. Keith G. Dumas P. Westhof E. RajBhandary U. Soll D. tRNA: Structure, Biosynthesis and Function. American Society for Microbiology, Washington, DC1995: 93-126Google Scholar). There is little detailed structural information on these tRNAs. No data are currently available that examine the structure of mammalian mitochondrial tRNAs with single nucleotide resolution. However, chemical and enzymatic probing has lead to the idea that these tRNAs have retained the basic cloverleaf structure of canonical tRNAs but that they lack several conserved tertiary interactions leading to a weaker three-dimensional structure (4Watanabe Y.-I. Kawai G. Yokogawa T. Hayashi N. Kumazawa Y. Ueda T. Nishikawa K. Hirao I. Miura K.-I. Watanabe K. Nucleic Acids Res. 1994; 22: 5378-5384Crossref PubMed Scopus (56) Google Scholar, 5Yokogawa T. Watanabe Y.-I. Kumazawa Y. Ueda T. Hirao I. Miura K.-I. Watanabe K. Nucleic Acids Res. 1991; 19: 6101-6105Crossref PubMed Scopus (68) Google Scholar, 6Wakita K. Watanabe W. Yokogawa T. Kumazawa Y. Nakamura S. Ueda T. Watanabe K. Nishikawa K. Nucleic Acids Res. 1994; 22: 347-353Crossref PubMed Scopus (75) Google Scholar, 7Helm M. Giege R. Florentz C. Biochemistry. 1999; 38: 13338-13346Crossref PubMed Scopus (174) Google Scholar, 8Helm M. Brule H. Friede D. Giege R. Putz D. Florentz C. RNA (N. Y.). 2000; 6: 1356-1379Crossref PubMed Scopus (242) Google Scholar). In particular, a number of the long range interactions between the D- and T-arms of the tRNAs appear to be missing.All 22 tRNAs that function in mammalian mitochondria are encoded in the mitochondrial DNA. Considerable interest in mitochondrial tRNAs centers on the occurrence of diseases arising from mutations in their genes that lead to maternally inherited genetic disorders (9Wittenhagen L.M. Kelley S.O. Trends Biochem. Sci. 2003; 28: 605-611Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10King M. Koga Y. Davidson M. Schon E. Mol. Cell. Biol. 1992; 12: 480-490Crossref PubMed Scopus (404) Google Scholar, 11Sternberg D. Chatzoglou E. Laforet P. Fayet G. Jardel C. Blondy P. Fardeau M. Amselem S. Eymard B. Lombes A. Brain. 2001; 124: 984-994Crossref PubMed Scopus (79) Google Scholar, 12Enriquez J. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (257) Google Scholar). The diseases associated with mitochondrial tRNA mutations may arise from failure in the processing of the tRNA (13Levinger L. Jacobs O. James M. Nucleic Acids Res. 2001; 29: 4334-4340Crossref PubMed Scopus (51) Google Scholar), from reduced stability of the tRNA (14Hao H. Moraes C.T. Mol. Cell. Biol. 1997; 17: 6831-6837Crossref PubMed Scopus (57) Google Scholar, 15Kelley S.O. Steinberg S.V. Schimmel P. Nat. Struct. Biol. 2000; 7: 862-865Crossref PubMed Scopus (54) Google Scholar), from a reduction in aminoacylation (12Enriquez J. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (257) Google Scholar, 16Ling J. Roy H. Qin D. Rubio M.A. Alfonzo J.D. Fredrick K. Ibba M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 15299-15304Crossref PubMed Scopus (38) Google Scholar, 17Cenatiempo Y. Deville F. Dondon J. Grunberg-Manago M. Sacerdot C. Hershey J.W. Hansen H.F. Petersen H.U. Clark B.F. Kjeldgaard M. la Cour T.F.M. Mortensen K.K. Nyborg J. Biochemistry. 1987; 26: 5070-5076Crossref PubMed Scopus (49) Google Scholar), from a reduced ability of the mutated aminoacyl-tRNA to interact with mitochondrial elongation factor Tu (EF-Tumt) 3The abbreviations used are: EF-Tumt, mitochondrial elongation factor Tu; hmtRNAMet, human mitochondrial tRNAMet; MetRS, methionyl-tRNA synthetase; hmMetRS, human mitochondrial methionyl-tRNA synthetase; MTF, methionyl-tRNA transformylase; SHAPE, selective 2′-hydroxyl acylation analyzed by primer extension; PMSF, phenylmethylsulfonyl fluoride; ;ME, ;-mercaptoethanol; 1M7, 1-methyl-7-nitroisatoic anhydride. 3The abbreviations used are: EF-Tumt, mitochondrial elongation factor Tu; hmtRNAMet, human mitochondrial tRNAMet; MetRS, methionyl-tRNA synthetase; hmMetRS, human mitochondrial methionyl-tRNA synthetase; MTF, methionyl-tRNA transformylase; SHAPE, selective 2′-hydroxyl acylation analyzed by primer extension; PMSF, phenylmethylsulfonyl fluoride; ;ME, ;-mercaptoethanol; 1M7, 1-methyl-7-nitroisatoic anhydride. (the corresponding prokaryotic factor is also designated EF1A) (16Ling J. Roy H. Qin D. Rubio M.A. Alfonzo J.D. Fredrick K. Ibba M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 15299-15304Crossref PubMed Scopus (38) Google Scholar), and from the failure of the tRNA to be correctly modified leading to translational defects (18Kirino Y. Yasukawa T. Ohta S. Akira S. Ishihara K. Watanabe K. Suzuki T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15070-15075Crossref PubMed Scopus (205) Google Scholar).Normally, protein biosynthetic systems have two tRNAMet species. One is used solely for initiation, and the other functions in polypeptide chain elongation. Animal mitochondria are quite unusual in that they contain a single gene for tRNAMet, which functions in both polypeptide chain initiation and chain elongation. As a result of this dual role, mitochondrial Met-tRNAMet must be recognized by the mitochondrial Met-tRNA transformylase (MTFmt) and be brought as fMet-tRNAMet to the ribosome for translational initiation (19Spencer A.C. Spremulli L.L. Nucleic Acids Res. 2004; 32: 5464-5470Crossref PubMed Scopus (50) Google Scholar). In addition, Met-tRNAMet must interact with elongation factor EF-Tumt and bind to the A-site of the ribosome during translational elongation. Thus, this tRNAMet is of central importance in mitochondrial translation.Human tRNAMet has a number of interesting features (Fig. 1A). The D-loop is somewhat small and lacks the G residues at positions 18 and 19 that facilitate interactions with the T-loop in the tertiary structure. The first position of the anticodon contains the rare modified base 5-formylcytidine. This modification may play a role in the unusual codon recognition requirements of this tRNA, which must recognize both AUG and AUA codons. The minor loop is short lacking the usual G47, and the T-stem has two adjacent pyrimidine:pyrimidine pairs (U-U and U-:). Furthermore, the T-loop lacks the T:C sequence and contains only six nucleotides instead of the normal seven. These unusual structural features suggest that human mitochondrial tRNAMet may have an intrinsically weak tertiary structure.Three interesting point mutations (T4409C, A4435G, and G4450A) occur in the gene for human tRNAMet (hmtRNAMet). The T4409C mutation (Fig. 1A) results in a U8 to C change at the corner of the acceptor stem and D-stem of hmtRNAMet. This mutation leads to mitochondrial myopathy resulting in dystrophic muscles and exercise intolerance (20Vissing J. Salamon M.B. Arlien-Soborg P. Norby S. Manta P. DiMauro S. Schmalbruch H. Neurology. 1998; 50: 1875-1878Crossref PubMed Scopus (59) Google Scholar). The A4435G mutation leads to the change of A37 to G37 in the anticodon loop of the tRNA (21Qu J. Li R. Zhou X. Tong Y. Lu F. Qian Y. Hu Y. Mo J.Q. West C.E. Guan M.X. Investig. Ophthalmol. Vis. Sci. 2006; 47: 475-483Crossref PubMed Scopus (119) Google Scholar). This mutation acts as a modulator of Leber's Hereditary Optic Neuropathy increasing the severity of this condition when it arises because of other mutations in the mitochondrial DNA. The G4450A mutation leads to loss of the final base pair in the T-stem (Fig. 1A). This mutation presents as splenic lymphoma, is largely confined to lymphocyte cells, and results in severely abnormal mitochondria leading to serious defects in energy production (22Lombes A. Bories D. Girodon E. Franchon P. Ngo M. Breton-Gorious J. Tulliez M. Goossens M. Hum. Mutat. 1998; 1: S175-S183Crossref PubMed Scopus (20) Google Scholar). A systematic examination of the structural and biochemical consequences of these mutations is lacking. Here we examine the structure of human mitochondrial tRNAMet and probe the effects of the 8U→C mutation on the structure and function of this tRNA.EXPERIMENTAL PROCEDURESRNA Synthesis—Human mitochondrial tRNAMet transcripts for aminoacylation experiments were prepared by in vitro transcription using the hammerhead ribozyme construct described previously (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar). The hmtRNAMet was purified by denaturing (10%) PAGE (29:1 acrylamide:bisacrylamide prepared with 7 m urea, 90 mm Tris borate, 2 mm EDTA), visualized by UV shadowing, excised from the gel, and recovered by passive elution in water followed by ethanol precipitation. hmtRNAMet transcripts for selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) experiments were prepared in the context of the structure cassette as described (24Wilkinson K.A. Merino E.J. Weeks K.M. Nat. Protoc. 2006; 1: 1610-1616Crossref PubMed Scopus (546) Google Scholar). D- and T-half-molecules were chemically synthesized (Dharmacon), purified, and analyzed as described previously (25Jones C. Spencer A.C. Hsu J. Spremulli L.L. Martinis S.A. DeRider M. Agris P.F. J. Mol. Biol. 2006; 362: 771-786Crossref PubMed Scopus (24) Google Scholar).Purification of E. coli Methionyl-tRNA Synthetase (MetRS) and Human Mitochondrial MetRS (hmMetRS)—A saturated overnight culture of JM109 cells carrying the pQE60-Escherichia coli MetRS plasmid construct (kindly provided by Uttam RajBhandary, Massachusetts Institute of Technology) was grown at 37 °C in 2× YT media (20 ml) supplemented with 50 ;g/ml ampicillin and used to inoculate 2 liters of 2×YT media (50 ;g/ml ampicillin). The cells were grown at 37 °C for 4 h (A600 = 0.6), induced with 50 ;m isopropyl ;-d-thiogalactopyranoside and then grown at 37 °C for 4 h post-induction. The cells were harvested by centrifugation at 4,000 rpm for 30 min. The cell pellet was resuspended in 500 ml of 10 mm Tris-HCl, pH 7.6, and then re-collected by low speed centrifugation. The cell pellet was fast frozen and stored at -80 °C until use.The cell pellet (7 g) was resuspended in 100 ml of lysis buffer (50 mm Tris-HCl, pH 7.6, 50 mm KCl, 10 mm MgCl2, 200 ;m phenylmethylsulfonyl fluoride (PMSF), 0.1% Triton X-100, and 7 mm ;-mercaptoethanol (;ME)) and sonicated on ice for 7 min with 10-s bursts followed by 50-s cooling periods. The cell lysate was centrifuged at 15,000 rpm for 30 min at 4 °C. E. coli MetRS was purified from the supernatant using 400 ;l of a 50% nickel-nitrilotriacetic acid (Qiagen) slurry in wash buffer (100 mm Tris-HCl, pH 7.6, 1 m KCl, 10 mm MgCl2, 10 mm imidazole, 200 ;m PMSF, and 7 mm ;ME). The resin was washed with 200 ml of wash buffer. The protein was eluted with 4 ml of elution buffer (100 mm Tris-HCl, pH 7.6, 50 mm KCl, 10 mm MgCl2, 150 mm imidazole, 200 ;m PMSF, and 7 mm ;ME). The protein sample was dialyzed against 2 volumes of 500 ml of dialysis buffer (50 mm Tris-HCl, pH 7.6, 50 mm KCl, 2.5 mm MgCl2, 0.1 mm EDTA, 10% glycerol and 7 mm ;ME) for 1 h.Cells carrying a plasmid encoding the His6-tagged human mitochondrial MetRS were grown as described (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar). The cells were lysed as described above, and the hmMetRS was further purified as described (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar).Purification of Bovine Mitochondrial Methionyl-tRNA Transformylase (MTFmt)—E. coli BL21 cells, carrying the pET15-bovine MTFmt plasmid construct, were grown as described (19Spencer A.C. Spremulli L.L. Nucleic Acids Res. 2004; 32: 5464-5470Crossref PubMed Scopus (50) Google Scholar). Cells were harvested and lysed as described for E. coli MetRS above. The protein was purified as described for the E. coli MetRS except that the buffers contained 50 mm Tris-HCl, pH 7.6. The purified protein sample was dialyzed against 2 volumes of 500 ml of MTF dialysis buffer (20 mm Tris-HCl, pH 7.6, 100 mm KCl, 10% glycerol, and 3 mm ;ME) for 1 h, fast-frozen, and stored at -80 °C.Assay for the Aminoacylation of Human Mitochondrial tRNAMet—The aminoacylation reactions for both the normal and 8U→C mutated tRNAMet transcripts were performed essentially as described (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar). Reaction mixtures (100 ;l) contained 50 mm Tris-HCl, pH 7.6, 2.5 mm MgCl2, 2.5 mm ATP, 0.2 mm spermine, 200 ;g/ml bovine serum albumin, 0.2 units/;l SUPERase·In RNase inhibitor, 40 ;m [35S]methionine (4,000 cpm/pmol), 50 nm human mitochondrial MetRS or 8 nm E. coli MetRS, and 1 ;m U8 or 8U→C hmtRNAMet. The amount of aminoacylated tRNA formed was determined by trichloroacetic acid-precipitable counts at the indicated times.Preparative Aminoacylation of [35S]Met-tRNAMet—Reaction mixtures (2 ml) were prepared as described above except that 20 ;m [35S]methionine (20,000 cpm/pmol), 0.5 ;m U8, or 8U→C hmtRNAMet, and saturating amounts of human mitochondrial MetRS were used. Reactions were incubated for 15 min at 37 °C, followed by phenol/chloroform extraction. The tRNAMet was collected by ethanol precipitation and then dissolved in 10 mm potassium succinate, pH 6.0, before use.Formylation of Human Mitochondrial Met-tRNAMet—Formylation reactions (5 ;l) contained 20 mm Tris-HCl, pH 7.6, 100 ;m EDTA, 150 mm KCl, 7 mm MgCl2, 10 mm ;ME, 125 ;m folinic acid (Sigma), 100 nm normal or 8U→C mutated [35S]Met-hmtRNAMet and 8 nm MTFmt. Reactions were performed at 37 °C for 0-8 min (0-min time point was taken in the absence of enzyme). At the indicated time, 83 mm NaOH (1 ;l of 500 mm) was added, and incubation was continued at 37 °C for 30 min. The [35S]Met and [35S]fMet in 5 ;l of each reaction were separated on Partisil LK5D TLC plates (Whatman) with a butanol:acetic acid:water (4:1:1) mixture. TLC plates were visualized by phosphorimaging (GE Healthcare) and the spots were analyzed using the ImageQuant program.Binding of Human Mitochondrial Met-tRNAMet to Bovine Mitochondrial EF-Tu (EF-Tumt)—EF-Tumt was prepared as described (26Bullard J.M. Cai Y.-C. Zhang Y. Spremulli L.L. Biochim. Biophys. Acta. 1999; 1446: 102-114Crossref PubMed Scopus (21) Google Scholar), except that the cells were lysed as described above for E. coli MetRS, and the high speed centrifugation step was omitted. Where indicated the normal U8 or 8U→C mutated hmtRNAs were phosphorylated with cold ATP using polynucleotide kinase (New England Biolabs) prior to large scale aminoacylation.To measure ternary complex formation, reaction mixtures (50 ;l) were prepared as reported (27Hunter S.E. Spremulli L.L. RNA Biol. 2004; 2: 95-102Crossref Scopus (8) Google Scholar) except that 20 mm Hepes-KOH, pH 7, and the indicated amounts of EF-Tumt were used. The reactions were incubated for 15 min at 0 °C or 6 min at 37 °C as indicated. Free [35S]Met-hmtRNAMet was digested by a 30-s incubation with 10 ;g of RNase A, and the reaction was terminated by the addition of cold 5% trichloroacetic acid. Following a 10-min incubation on ice, the [35S]Met-hmtRNAMet precipitate was collected on nitrocellulose filters and quantified by liquid scintillation counting. Determination of the Kd for ternary complex formation was carried out as described previously (28Cai Y.-C. Bullard J.M. Thompson N.L. Spremulli L.L. J. Biol. Chem. 2000; 275: 20308-20314Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar).Degradation of hmtRNAMet in a Mitochondrial Extract—Bovine mitoplasts (0.2 g) were prepared as described (29Schwartzbach C. Farwell M. Liao H.-X. Spremulli L.L. Methods Enzymol. 1996; 264: 248-261Crossref PubMed Google Scholar). Mitoplasts were lysed in buffer (2 ml) containing 15 mm Tris-HCl, pH 7.6, 40 mm KCl, 6 mm MgCl2, 6 mm ;ME, 0.8 mm EDTA, and 1.6% Triton X-100 by hand homogenization. The extract was clarified by centrifugation at 10,000 rpm for 15 min at 4 °C. Normal U8 and 8U→C mutated hmtRNAMet were 32P-end-labeled using polynucleotide kinase (New England Biolabs) according to the manufacturer's instructions. Reaction mixtures (10 ;l) contained 100 nm normal U8 or 8U→C mutated [32P]hmtRNAMet and the indicated amount of the extract in the lysis buffer above. Incubation was for 10 min at 37 °C, and cold trichloroacetic acid precipitation was used to quantitate the amount of [32P]hmtRNAMet remaining.Selective 2′-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE) Analysis of Normal U8 and 8U→C Mutated hmtRNAMet Transcripts—Normal U8 or 8U→C mutated hmtRNAMet (12 pmol, 0.33 ;m) in 36 ;l of nuclease-free water (Ambion) was incubated at 50 °C for 2 min and then cooled on ice for 2 min. The RNA was divided into 2 aliquots of 4 pmol (12 ;l) and 8 pmol (24 ;l). Folding buffer (6 ;l; 333 mm Hepes-KOH, pH 8, 333 mm NaCl) was added to the 4 pmol of RNA, and folding buffer with 20 mm Mg2+ (12 ;l) was added to the 8 pmol of RNA, and the two samples were incubated at 37 °C for 20 min. To 1 ;l of 100 mm 1-methyl-7-nitroisotoic anhydride (1M7) in anhydrous DMSO or 1 ;l of anhydrous DMSO (control), 9 ;l (2 pmol) of folded RNA was added and allowed to react at 37 °C for 70 s (5 half-lives). The balance of the RNA folded in the presence of Mg2+ (18 ;l) was divided into 2 aliquots of 2 pmol (9 ;l) each and stored at 37 °C for sequencing. MgCl2 (1 ;l; 64 mm) was added to the RNA treated with the folding buffer in the absence of Mg2+. Radiolabeled oligonucleotide (0.3 ;m;3 ;l; 5′-32P-GAACCGGACCGAAGCCCG, obtained from the Nucleic Acids Core Facility at University of North Carolina) was added to the 1M7-treated, DMSO-treated, or untreated RNA (2 pmol), and the samples were incubated at 65 °C for 5 min and then at 35 °C for 20 min for primer annealing. To each reaction, reverse transcription buffer (6 ;l; 250 mm KCl, 167 mm Tris-HCl, pH 8.3, 17 mm dithiothreitol, and 0.42 mm each dNTP) was added. Then either ddCTP or ddTTP (1 ;l; 5 mm; Amersham Biosciences) was added to the untreated RNA. After heating to 52 °C, reverse transcriptase (1 ;l; 200 units; Superscript III, Invitrogen) was added, and the primer extension reactions were performed at 52 °C for 5 min. Reactions were quenched with 4 m NaOH (1 ;l) and heated at 95 °C for 5 min. For gel analysis, a gel loading solution (29 ;l; 138 mm unbuffered Tris-HCl, 73% (v/v) formamide, 2 mm Tris borate, 86 mm EDTA, pH 8, with xylene cyanol and bromphenol blue) was added, and the samples were heated at 95 °C for an additional 5 min. The cDNA products from the + and - 1M7 and sequencing reactions were separated by denaturing gel electrophoresis (10% polyacrylamide). Samples on gels (21 cm × 40 cm × 0.4 mm) were subjected to electrophoresis at 1400 V for ∼2.5 h. Gels were visualized by phosphorimaging (GE Healthcare). The + and - 1M7 band intensities were quantified using SAFA (30Das R. Laederach A. Pearlman S.M. Herschlag D. Altman R.B. RNA (N. Y.). 2005; 11: 344-354Crossref PubMed Scopus (254) Google Scholar) and corrected for signal drop-off (31Badorrek C.S. Weeks K.M. Biochemistry. 2006; 45: 12664-12672Crossref PubMed Scopus (37) Google Scholar). SHAPE reactivities were normalized by subtracting intensities for the -1M7 control from the +1M7 reaction and dividing each by the average reactivity of the most reactive 7% of the nucleotides. To facilitate comparison of the normal U8 and 8U→C mutant tRNAs, the two data sets were normalized to the reactivity of the -CCA end nucleotides. The reactivity of each nucleotide was assigned a value between 0 and 1. Nucleotides fall into one of four categories (32Wilkinson K.A. Merino E.J. Weeks K.M. J. Am. Chem. Soc. 2005; 127: 4659-4667Crossref PubMed Scopus (125) Google Scholar, 33Merino E.J. Wilkinson K.A. Coughlan J.L. Weeks K.M. J. Am. Chem. Soc. 2005; 127: 4223-4231Crossref PubMed Scopus (551) Google Scholar) as follows: unreactive (0.000-0.055), low reactivity (0.055-0.110), moderately reactive (0.110-0.220), or highly reactive (0.220-1.000).Structural Studies of hmtRNAMet Half-molecules—Reconstitution of hmtRNAMet from U8 and 8U→C D-half-molecules with T-half-molecules required Mg2+ and was assessed by gel mobility shift assays using native 15% PAGE in Tris borate buffer (89 mm Tris base, 89 mm boric acid, pH 8.3) with and without 3 mm Mg2+ at 4 °C (25Jones C. Spencer A.C. Hsu J. Spremulli L.L. Martinis S.A. DeRider M. Agris P.F. J. Mol. Biol. 2006; 362: 771-786Crossref PubMed Scopus (24) Google Scholar). The concentration of the D-half-molecule was held constant at 31.2 ;m, whereas the concentration of the T-half-molecule was varied from 4.2 to 112 ;m.UV-Monitored Thermodynamic Experiments—The half-molecule RNA samples were dissolved in the above Tris borate buffer used for the PAGE experiments to obtain an RNA concentration of 1.2 ;m. MgCl2 was added to a concentration of 3 mm. UV-monitored thermal denaturations and re-naturations were replicated 10 times and monitored by measuring UV absorbance (260 nm) using a Cary 3 spectrophotometer as published (34Ashraf S.S. Guenther R.H. Ansari G. Malkiewicz A. Sochacka E. Agris P.F. Cell Biochem. Biophys. 2000; 33: 241-252Crossref PubMed Scopus (30) Google Scholar, 35Yarian C.S. Basti M.M. Cain R.J. Ansari G. Guenther R.H. Sochacka E. Czerwinska G. Malkiewicz A. Agris P.F. Nucleic Acids Res. 1999; 27: 3543-3549Crossref PubMed Scopus (88) Google Scholar). The data points were averaged over 20 s and recorded with a temperature change of 1 °C per min from 4 to 90 °C. The one most inconsistent of the 10 melting transitions (either a denaturation or renaturation) was discarded from each set, and the resulting data were averaged on a point-by-point basis.For UV melts of the hmtRNAMet transcripts, the normal U8 and the 8U→C hmtRNAMet were dialyzed against water using 10-kDa cutoff dialysis cups (Stratagene). The U8 and 8U→C hmtRNAMet transcripts were diluted to 0.5 ;m in a buffer containing 10 mm NaCl and 10 mm Hepes-KOH, pH 8.0. The thermal denaturation of the tRNAs was monitored by UV absorbance at 260 nm using a Cary 3 spectrophotometer. Data points were recorded once per min from 4 to 95 °C with a temperature change of 1 °C per min. Following thermal renaturation, 6 mm Mg2+ was added to the U8 and 8U→C transcripts, and the UV-monitored thermal denaturation experiments were repeated.RESULTSAminoacylation of the Normal and 8U→C Mutated tRNAMet—Previous studies (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar) have shown that the transcript of mitochondrial tRNAMet has aminoacylation properties similar to those observed with the native tRNA. Thus, it was possible to use the normal transcript and a transcript containing the 8U→C mutation for studies on the effect of the mutation on the properties of the tRNA. The 8U→C mutation leads to a myopathy presumably arising from a reduction in translational activity in mitochondria. To determine the biochemical consequence of the 8U→C mutation, the abilities of the U8 and 8U→C hmtRNAMet transcripts to be aminoacylated by the human mitochondrial methionyl-tRNA synthetase (hmMetRS) were tested. Aminoacylation is an early step required for the tRNA to be used in either the elongation or initiation phase of protein synthesis and is thus of central importance for protein synthesis in mitochondria. The normal U8 transcript was aminoacylated as expected (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar); however, the 8U→C mutation caused a significant reduction in the rate of aminoacylation of the tRNA by hmMetRS (Fig. 1B). This observation provides one clear rationale for the failure of this tRNA to function in mitochondrial protein biosynthesis.Not unexpectedly, the normal hmtRNAMet was aminoacylated by the E. coli MetRS (Fig. 1C). Interestingly, whereas the 8U→C hmtRNAMet was poorly aminoacylated by the hmMetRS, it was not aminoacylated at all by the E. coli MetRS (Fig. 1C) suggesting that the mutated tRNA had a significantly altered structure. The hmMetRS is believed to be both structurally and functionally homologous to its prokaryotic counter-part (23Spencer A.C. Heck A.H. Takeuchi N. Watanabe K. Spremulli L.L. Biochemistry. 2004; 43: 9743-9754Crossref PubMed Scopus (38) Google Scholar). However, this work demonstrates that the hmMetRS is less discriminatory than E. coli MetRS for the structure of the tRNA. Because a major determinant in the recognition of tRNAMet by the MetRS is thought to lie in the anticodon sequence that is unchanged (36Schulman L.H. Pelka H. Science. 1988; 242: 765-768Crossref PubMed Scopus (184) Google Scholar), the weak aminoacylation most likely reflects significant structural alterations in the tRNA as a result of the mutation.Formylation of Normal and 8U→C Mutated Met-tRNAMet—The defective aminoacylation of the 8U→C hmtRNAMet made it difficult to assess the effects of the mutation on additional steps in protein biosynthesis. However, small amounts of the aminoacylated 8U→C mutated hmtRNAMet could be isolated, permitting a limited investigation of additional steps in translation.In the mammalian mitochondrial system, the Met-tRNAMet must be formylated by the mitochondrial transformylase (MTFmt) to be used in initiation (19Spencer A.C. Spremulli L.L. Nucleic Acids Res. 2004; 32: 5464-5470Crossref PubMed Scopus (50) Google Scholar, 37Takeuchi N. Kawakami M. Omori A. Ueda T. Spremulli L.L. Watanabe K. J. Biol. Chem. 1998; 273: 15085-15090Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The abilities of the U8 and 8U→C Met-tRNAMet to be formylated were tested by incubation of the [35S]Met-tRNA with th}, 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 tRNA(Glu) 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 (mnm(5)s(2)U(34)) in the first or wobble anticodon position of E. coli tRNA(Glu). 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 (ASL(Glu)-s(2)U(34)) and without s(2)U(34) are bound by ERS and inhibit aminoacylation of the native tRNA. ASL(Glu), ASL(Glu)-s(2)U(34), and a minihelix(Glu) composed of identity determinants of the amino acid accepting stem were thermally stable under conditions of aminoacylation (T(m)s = 75 +/- 1.5, 76 +/- 0.9 and 83 +/- 2.0 degrees C, respectively). In binding competition, the modified ASL(Glu)-s(2)U(34) bound ERS with a higher affinity (half maximal inhibiting concentration, IC(50), 5.1 +/- 0.4 microM) than its unmodified counterpart, ASL(Glu) (IC(50), 10.3 +/- 0.6 microM). The minihelix(Glu), ASL(Glu)-s(2)U(34) and ASL(Glu) bound ERS with K(d)s of 9.9 +/- 3.3, 6.5 +/- 1.7 and 20.5 +/- 3.8 microM. ERS aminoacylation of tRNA(Glu) was inhibited by the tRNA fragments. Unmodified ASL(Glu), minihelix(Glu), and ASL(Glu)-s(2)U(34) exhibited a K(ic) of 1.9 +/- 0.2 microM, 4.1 +/- 0.2 microM, and 6.5 +/- 0.1 microM, respectively. The modified ASL(Glu)-s(2)U(34), 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}, 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 K d s 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 tRNA Gly (ASL Gly GCC ) specifically and with a significant affinity ( K d = 20.2 ± 1.4 μM). Thus, the bimolecular 5′-UTR and ASL Gly GCC 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}, 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 Ka of 5.3 × 104 M–1. Ka 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}, 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 tRNAUUULys 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 TΨC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TΨC domain, rT54, Ψ55 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 tRNAPhe 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 Kds (rT54 = 6 ± 2, m5C49 = 11 ± 2, Ψ55 = 14 ± 5, and rT54,Ψ55 = 11 ± 3 µM) significantly lower than that of the unmodified T‐half (40 ± 10 µM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNAPhe and native yeast tRNAPhe, as determined by lead cleavage patterns at U17 and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA’s TΨC 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 tRNALys3 anticodon stem and loop domain (ASLLys3) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t6A37) to tRNALys3 functions. The t6A37-modified anticodon stem and loop domain of tRNALys3UUU (ASLLys3UUU- t6A37) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASLLys3UUU 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 Å (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 ASLLys3UUU-t6A37 loop is significantly different than that of the unmodified ASLLys3UUU, although the five canonical base pairs of both ASLLys3UUU stems are in the standard A-form of helical RNA. t6A37, 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 U33·A37 base pair that is observed in the unmodified ASLLys3UUU. The anticodon wobble position U34 nucleobase in ASLLys3UUU-t6A37 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 ASLLys3UUU tendency to prematurely terminate translation and −1 frame shift. At the pH 5.6 conditions of our structure determination, A38 is protonated and positively charged in ASLLys3UUU-t6A37 and the unmodified ASLLys3UUU. The ionized carboxylic acid moiety of t6A37 possibly neutralizes the positive charge of A+38. The protonated A+38 can base pair with C32, but t6A37 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 ASLLys3UUU would bind as well as ASLLys3UUU-t6A37. t6A37 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 TΨC stem and loop (TSL) of tRNA contains highly conserved nucleoside modifications, m5C49, T54, Ψ55 and m1A58. U54 is methylated to m5U (T) by m5U54 methyltransferase (RUMT); A58 is methylated to m1A by m1A58 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 (T54 and Ψ55), naturally occurring modifications at unnatural positions (m5C60), altered sugar puckers (dU54 and/or dU55) or with disrupted U-turn interactions (m1Ψ55 or m1m3Ψ55). The unmodified heptadecamer TSL was a substrate of both RAMT and RUMT. The presence of T54 increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate. Local conformation around U54 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}, 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 TΨC 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 TΨC 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}, 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 tRNAPhe 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 tRNAPhe, 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 tRNAPhe enhanced protection of C1400, but the ASL-U33 and U33-substituted ASLs did not. Two residues, G926 and G1338 with Kps ≈ 50–60 nM, were afforded significantly greater protection by both yeast tRNAPhe and the ASL-U33 than other residues, such as A532, A794, C795, and A1339 (Kps ≈ 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} }