Modified Uridines with C5-methylene Substituents at the First Position of the tRNA Anticodon Stabilize U·G Wobble Pairing during Decoding
2008; Elsevier BV; Volume: 283; Issue: 27 Linguagem: Inglês
10.1074/jbc.m800233200
ISSN1083-351X
AutoresShinya Kurata, Albert Weixlbaumer, Takashi Ohtsuki, Tomomi Shimazaki, Takeshi Wada, Yohei Kirino, Kazuyuki Takai, Kimitsuna Watanabe, V. Ramakrishnan, Tsutomu Suzuki,
Tópico(s)Genomics and Phylogenetic Studies
ResumoPost-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm5U) at the wobble position. However, the functional properties of the C5-substituents of xm5U in codon recognition remain elusive. We previously found that mitochondrial tRNAsLeu(UUR) with pathogenic point mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (τm5U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAsLeu(UUR) with or without xm5U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNAArg lacking mnm5U modification in a knock-out strain of the modifying enzyme (ΔmnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm5U modification plays a critical role in decoding NNG codons by stabilizing U·G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing τm5U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the τm5U·G base pair does not have classical U·G wobble geometry. These structures provide help to explain how the τm5U modification enables efficient decoding of UUG codons. Post-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm5U) at the wobble position. However, the functional properties of the C5-substituents of xm5U in codon recognition remain elusive. We previously found that mitochondrial tRNAsLeu(UUR) with pathogenic point mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (τm5U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAsLeu(UUR) with or without xm5U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNAArg lacking mnm5U modification in a knock-out strain of the modifying enzyme (ΔmnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm5U modification plays a critical role in decoding NNG codons by stabilizing U·G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing τm5U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the τm5U·G base pair does not have classical U·G wobble geometry. These structures provide help to explain how the τm5U modification enables efficient decoding of UUG codons. The genetic code is deciphered by the anticodons of tRNAs, which carry an amino acid at the 3′ end, bind to a specific codon in the mRNA, and transfer their amino acid to the growing polypeptide chain on the ribosome. In codon-anticodon interactions in the ribosome, the second and third bases (positions 35 and 36) of the anticodon base pair with the second and first bases of the codon, respectively, following Watson-Crick (WC) 2The abbreviations used are: WC, Watson-Crick; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; cmnm5U, 5-carboxymethylaminomethyluridine; τm5U, 5-taurinomethyluridine; mt, mitochondrial; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid; ASL, anticodon stem loop; xm5U, 5-methyluridine derivative; xm5Um, 5-methyl-2′-O-methyl derivative; mnm5U, 5-methylaminomethyluridine; mnm5s2U, 5-methylaminomethyl-2-thiouridine; mcm5U, 5-methoxycarbonylmethyluridine; mcm5Um, 5-methoxycarbonylmethyl-2′-O-methyluridine. -type pairing rules. Structural studies of the 30 S ribosomal subunit revealed that the conserved bases A1492, A1493, and G530 in the decoding center of the 16 S rRNA specifically monitor these two WC-type pairings by A-minor interactions (1Ogle J.M. Brodersen D.E. Clemons Jr., W.M. Tarry M.J. Carter A.P. Ramakrishnan V. Science. 2001; 292: 897-902Crossref PubMed Scopus (967) Google Scholar, 2Ogle J.M. Murphy F.V. Tarry M.J. Ramakrishnan V. Cell. 2002; 111: 721-732Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar). These interactions induce a large conformational rearrangement of the 30 S subunit that is necessary for tRNA selection and maintaining decoding fidelity. In contrast, base-pairing between the first base of the anticodon (position 34) and the third base of the codon does not always conform to WC-type pairing rules, so that synonymous codons for an amino acid are deciphered by a minimum set of tRNA anticodons. This behavior is referred to as "wobble pairing" (3Crick F.H. J. Mol. Biol. 1966; 19: 548-555Crossref PubMed Scopus (1305) Google Scholar), which is a highly evolved system required for the degeneracy of the genetic code by which 61 sense codons are deciphered into 20 amino acids by a limited set of tRNA species. Modified nucleosides are often found at the wobble position of tRNA anticodons (4Bjork G.R. Biosynthesis and Function of Modified Nucleosides. American Society for Microbiology Press, Washington, DC1995: 165-205Google Scholar, 5Curran J.F. Modified Nucleosides in Translation. American Society for Microbiology Press, Washington, DC1998: 493-516Google Scholar, 6Yokoyama S. Nishimura S. Soll D. Rajbandary U.L. Modified Nucleosides and Codon Recognition. American Society for Microbiology Press, Washington, DC1995: 207-224Google Scholar, 7Suzuki T. Fine-tuning of RNA Functions by Modification and Editing. Springer-Verlag New York Inc, New York2005: 24-69Google Scholar, 8Agris P.F. Vendeix F.A. Graham W.D. J. Mol. Biol. 2007; 366: 1-13Crossref PubMed Scopus (409) Google Scholar). The wobble modifications play critical roles in modulating codon recognition by restricting, expanding, or altering the decoding properties of the tRNAs (7Suzuki T. Fine-tuning of RNA Functions by Modification and Editing. Springer-Verlag New York Inc, New York2005: 24-69Google Scholar). In contrast to the first and second codon-anticodon base pair, the ribosome imposes less restraints on the wobble base pair (1Ogle J.M. Brodersen D.E. Clemons Jr., W.M. Tarry M.J. Carter A.P. Ramakrishnan V. Science. 2001; 292: 897-902Crossref PubMed Scopus (967) Google Scholar), so that various wobble base pair geometries as well as modifications can readily be accommodated in the decoding center. According to the original wobble rule (3Crick F.H. J. Mol. Biol. 1966; 19: 548-555Crossref PubMed Scopus (1305) Google Scholar), unmodified uridine at the wobble position (U34) was proposed to recognize only A and G at the third codon position. However, U34 can actually base-pair with any of the four bases due to its conformational flexibility (four-way wobbling). In fact, U34 is frequently found in tRNA species that are responsible for entire codon family boxes, in which four codons are synonymous, from Mycoplasma spp. and mitochondria (9Bonitz S.G. Berlani R. Coruzzi G. Li M. Macino G. Nobrega F.G. Nobrega M.P. Thalenfeld B.E. Tzagoloff A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3167-3170Crossref PubMed Scopus (220) Google Scholar, 10Barrell B.G. Anderson S. Bankier A.T. de Bruijn M.H. Chen E. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3164-3166Crossref PubMed Scopus (230) Google Scholar, 11Andachi Y. Yamao F. Iwami M. Muto A. Osawa S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7398-7402Crossref PubMed Scopus (39) Google Scholar, 12Inagaki Y. Kojima A. Bessho Y. Hori H. Ohama T. Osawa S. J. Mol. Biol. 1995; 251: 486-492Crossref PubMed Scopus (51) Google Scholar). In bacteria and eukaryotic cytoplasm, uridines at the wobble position of tRNAs are often post-transcriptionally modified (4Bjork G.R. Biosynthesis and Function of Modified Nucleosides. American Society for Microbiology Press, Washington, DC1995: 165-205Google Scholar, 7Suzuki T. Fine-tuning of RNA Functions by Modification and Editing. Springer-Verlag New York Inc, New York2005: 24-69Google Scholar). These modifications are classified into two groups according to their distinct chemical structures and decoding properties: 5-hydroxyuridine derivatives (xo5U) with an oxygen atom directly bonded to the C5 atom of the uracil base and 5-methyluridine derivatives (xm5U) with a methylene carbon directly bonded to the C5 atom. The xo5U type modifications are often found in bacterial tRNAs that are responsible for family boxes. Genetic and biochemical studies revealed that, in E. coli tRNAs, 5-carboxymethoxyuridine (cmo5U) is required to read A, G, and U efficiently in vitro (13Samuelsson T. Elias P. Lustig F. Axberg T. Folsch G. Akesson B. Lagerkvist U. J. Biol. Chem. 1980; 255: 4583-4588Abstract Full Text PDF PubMed Google Scholar, 14Phelps S.S. Malkiewicz A. Agris P.F. Joseph S. J. Mol. Biol. 2004; 338: 439-444Crossref PubMed Scopus (53) Google Scholar) and to recognize all four bases in vivo in a mutant strain lacking other isoacceptors (15Nasvall S.J. Chen P. Bjork G.R. RNA. 2004; 10: 1662-1673Crossref PubMed Scopus (91) Google Scholar, 16Sorensen M.A. Elf J. Bouakaz E. Tenson T. Sanyal S. Bjork G.R. Ehrenberg M. J. Mol. Biol. 2005; 354: 16-24Crossref PubMed Scopus (43) Google Scholar). In contrast, xm5U type modifications are found at the wobble position of tRNAs that are responsible for purine-ending split codon boxes (NNR) (7Suzuki T. Fine-tuning of RNA Functions by Modification and Editing. Springer-Verlag New York Inc, New York2005: 24-69Google Scholar). The xm5U type modifications include 2-thiouridine derivatives (xm5s2U) and 2′-O-methyluridine derivatives (xm5Um). 5-Methylaminomethyluridine (mnm5U) and its 2-thio derivative (mnm5s2U) are typical xm5U type modifications found in bacterial tRNAs, whereas 5-methoxycarbonylmethyluridine (mcm5U) and its 2-thio (mcm5s2U) and 2′-O-methyl derivatives (mcm5Um) are found only in eukaryotic tRNAs. 5-Carboxymethylaminomethyluridine (cmnm5U) (see Fig. 1A) can be found at the wobble position of tRNAs from Mycoplasma spp. and yeast mitochondria (17Martin R.P. Sibler A.P. Gehrke C.W. Kuo K. Edmonds C.G. McCloskey J.A. Dirheimer G. Biochemistry. 1990; 29: 956-959Crossref PubMed Scopus (34) Google Scholar, 18Andachi Y. Yamao F. Muto A. Osawa S. J. Mol. Biol. 1989; 209: 37-54Crossref PubMed Scopus (151) Google Scholar). In bacteria, cmnm5(s2)U is a modification intermediate of mnm5(s2)U, but cmnm5s2U and cmnm5Um are also found at the wobble positions of E. coli tRNALeu4 and tRNAGln1, respectively. 3K. Miyauchi and T. Suzuki unpublished result. The conformation of xm5s2U is largely fixed in the C3′-endo form of ribose puckering due to the large van der Waals radius of the 2-thio atom causing steric repulsion of the 2′-oxygen atom (19Yokoyama S. Watanabe T. Murao K. Ishikura H. Yamaizumi Z. Nishimura S. Miyazawa T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4905-4909Crossref PubMed Scopus (235) Google Scholar). Due to its conformational rigidity, the xm5s2U modification prefers to base-pair with A and prevents misreading of NNY codons (6Yokoyama S. Nishimura S. Soll D. Rajbandary U.L. Modified Nucleosides and Codon Recognition. American Society for Microbiology Press, Washington, DC1995: 207-224Google Scholar, 19Yokoyama S. Watanabe T. Murao K. Ishikura H. Yamaizumi Z. Nishimura S. Miyazawa T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4905-4909Crossref PubMed Scopus (235) Google Scholar), regardless of the chemical characteristics of the C5-substituents. Compared with the 2-thio group of xm5s2U, the chemical nature and functional roles of C5-substituents of xm5U remain elusive. The mammalian mitochondrial decoding system utilizes a limited set of tRNAs (22 species) that are capable of deciphering the 60 sense codons in the 13 protein genes encoded in mitochondrial (mt) DNA (10Barrell B.G. Anderson S. Bankier A.T. de Bruijn M.H. Chen E. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3164-3166Crossref PubMed Scopus (230) Google Scholar, 20Watanabe K. Osawa S. tRNA Sequence and Variations in the Genetic Code. American Society for Microbiology Press, Washington, DC1995: 225-250Google Scholar). The wobble modifications play an essential role in this decoding system. The four-way wobble rule of U34 reduces the total number of tRNA species required. In fact, each family box of codons is deciphered by a single tRNA with an unmodified wobble uridine. In human (and bovine) mt tRNAs responsible for decoding purine-ending two-codon sets, we previously identified two novel xm5U wobble modifications that possess a sulfonic acid group derived from taurine: 5-taurinomethyluridine (τm5U) (see Fig. 1A) in tRNAs for Leu(UUR) and Trp and 5-taurinomethyl-2-thiouridine (τm5s2U) in tRNAs for Lys, Gln, and Glu (21Suzuki T. Suzuki T. Wada T. Saigo K. Watanabe K. EMBO J. 2002; 21: 6581-6589Crossref PubMed Scopus (280) Google Scholar). 4T. Suzuki and T. Suzuki, unpublished results. These taurine-containing uridines are synthesized by direct incorporation of dietary taurine, indicating that taurine is a constituent of biological macromolecules and that there is a catabolic flow of intracellular taurine into mitochondria. Previously, we reported that the τm5(s2)U modifications are defective in mutant tRNAs from cells harboring mitochondrial encephalomyopathies (22Yasukawa T. Suzuki T. Suzuki T. Ueda T. Ohta S. Watanabe K. J. Biol. Chem. 2000; 275: 4251-4257Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 23Yasukawa T. Suzuki T. Ishii N. Ueda T. Ohta S. Watanabe K. FEBS Lett. 2000; 467: 175-178Crossref PubMed Scopus (115) Google Scholar, 24Kirino Y. Goto Y. Campos Y. Arenas J. Suzuki T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7127-7132Crossref PubMed Scopus (130) Google Scholar, 25Kirino Y. Suzuki T. RNA Biol. 2005; 2: 41-44Crossref PubMed Scopus (66) Google Scholar). The mutant A8344G mt tRNALys from MERRF (myoclonus epilepsy associated with ragged red fibers) patients possesses an unmodified wobble uridine instead of the normal τm5s2U modification (23Yasukawa T. Suzuki T. Ishii N. Ueda T. Ohta S. Watanabe K. FEBS Lett. 2000; 467: 175-178Crossref PubMed Scopus (115) Google Scholar, 26Yasukawa T. Kirino Y. Ishii N. Holt I.J. Jacobs H.T. Makifuchi T. Fukuhara N. Ohta S. Suzuki T. Watanabe K. FEBS Lett. 2005; 579: 2948-2952Crossref PubMed Scopus (65) Google Scholar). In one of five pathogenic mutations associated with MELAS, a mutant mt tRNALeu(UUR) also lacks the normal τm5U modification (22Yasukawa T. Suzuki T. Suzuki T. Ueda T. Ohta S. Watanabe K. J. Biol. Chem. 2000; 275: 4251-4257Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 24Kirino Y. Goto Y. Campos Y. Arenas J. Suzuki T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7127-7132Crossref PubMed Scopus (130) Google Scholar). Biochemical studies using an in vitro mitochondrial translation system revealed that the wild type tRNALeu(UUR) whose τm5U modification was surgically replaced by an unmodified uridine exhibited severely reduced UUG decoding but no decrease in UUA decoding (27Kirino 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 (211) Google Scholar). This finding strongly suggests that a UUG codon-specific translational defect of mutant mt tRNALeu(UUR) lacking the wobble modification is the primary cause of MELAS at the molecular level. In this study, we examined the decoding properties of cmnm5U- and τm5U-modified tRNAs using an E. coli in vitro translation system to elucidate the functional roles played by xm5U modifications. We chose to compare the cmnm5U modification with τm5U because cmnm5U contains glycine-derived substituents, the chemical characteristics of which are similar to the taurine-derived substituent of τm5U. The decoding properties of the xm5U modification were also investigated in cells of the knock-out strain for the modification enzyme mnmE, which generates the xm5U modification. Finally, we directly observed by crystallography the nature of the τm5U·G base pair at the wobble position at the ribosomal A-site. Our results demonstrate the critical role played by xm5U modifications in decoding NNG codons. Construction of E. coli tRNALeu(UUR) Bearing the Wobble Modification—The cmnm5U and τm5U nucleosides were chemically synthesized (21Suzuki T. Suzuki T. Wada T. Saigo K. Watanabe K. EMBO J. 2002; 21: 6581-6589Crossref PubMed Scopus (280) Google Scholar, 28Malkiewicz A. Sochacka E. Sayed Ahmed A.F. Yassin S. Tetrahedron Lett. 1983; 24: 5395-5398Crossref Scopus (14) Google Scholar, 29Murao K. Ishikura H. Nucleic Acids Res. Special Publication. 1978; 5: s333-s336Crossref Scopus (15) Google Scholar), and 5′- and 3′(2′)-diphosphorylation of these nucleosides was performed as previously described (30Barrio J.R. Barrio M.C. Leonard N.J. England T.E. Uhlenbeck O.C. Biochemistry. 1978; 17: 2077-2081Crossref PubMed Scopus (77) Google Scholar). The protocol for tRNA construction by molecular surgery is outlined in Fig. 1C and was carried out as described previously (31Kurata S. Ohtsuki T. Suzuki T. Watanabe K. Nucleic Acids Res. 2003; 31: e145Crossref PubMed Scopus (17) Google Scholar, 32Ohtsuki T. Kawai G. Watanabe K. J. Biochem. (Tokyo). 1998; 124: 28-34Crossref PubMed Scopus (28) Google Scholar, 33Suzuki T. Ueda T. Watanabe K. EMBO J. 1997; 16: 1122-1134Crossref PubMed Scopus (102) Google Scholar), with slight modifications. pcmnm5Up or pτm5Up was ligated to the 3′-end of the 5′-fragment (5′-HO-GGCCGGAUGGUGGAAUCGGUAGACACAAGGGAUU-OH-3′) at 11 °C for 16 h in a reaction mixture containing 50 mm Tris-HCl (pH 7.6), 15 mm MgCl2, 3.5 mm DTT, 15 μg/ml bovine serum albumin, 5% polyethylene glycol, 300 μm ATP, 1.8 mm pcmnm5Up or pτm5Up, 90 μm 5′-fragment, and 1.6 units/μl T4 RNA ligase. Subsequently, periodate oxidation was performed to inactivate the unligated substrate (31Kurata S. Ohtsuki T. Suzuki T. Watanabe K. Nucleic Acids Res. 2003; 31: e145Crossref PubMed Scopus (17) Google Scholar). Sodium periodate (NaIO4) was added to the reaction mixture to a final concentration of 10 mm, and the solution was incubated on ice for 40 min in the dark. Sodium periodate was removed from the RNAs by ethanol precipitation. The 3′-phosphate of the ligated 5′-fragment was then removed using bacterial alkaline phosphatase, as described (32Ohtsuki T. Kawai G. Watanabe K. J. Biochem. (Tokyo). 1998; 124: 28-34Crossref PubMed Scopus (28) Google Scholar). The ligated 5′-fragment and the 3′-fragment (5′-HO-AAAAUCCCUCGGCGUUCGCGCUGUGCGGGUUCAAGUCCCGCUCCGGCUACCA-OH-3′) (90 μm each) were mixed and heated at 65 °C for 7 min, annealed at room temperature for 1 h in 50 mm Tris-HCl (pH 7.6) and 15 mm MgCl2, and then ligated at 37 °C for 1 h in buffer containing 60 mm Tris-HCl (pH 7.6), 17.5 mm MgCl2, 3.5 mm DTT, 10 μg/ml bovine serum albumin, 300 μm ATP, and 1.6 units/μl T4 RNA ligase. Subsequently, the 5′ termini of the tRNAs were phosphorylated (31Kurata S. Ohtsuki T. Suzuki T. Watanabe K. Nucleic Acids Res. 2003; 31: e145Crossref PubMed Scopus (17) Google Scholar). The resultant tRNAs were purified by separation on 10% denaturing PAGE. The sequences of the tRNAs were confirmed enzymatically (34Donis-Keller H. Nucleic Acids Res. 1980; 8: 3133-3142Crossref PubMed Scopus (258) Google Scholar) and by mass spectrometry fragment analysis (35Noma A. Kirino Y. Ikeuchi Y. Suzuki T. EMBO J. 2006; 25: 2142-2154Crossref PubMed Scopus (159) Google Scholar, 36Suzuki T. Ikeuchi Y. Noma A. Suzuki T. Sakaguchi Y. Methods Enzymol. 2007; 425: 211-229Crossref PubMed Scopus (100) Google Scholar, 37Kaneko T. Suzuki T. Kapushoc S.T. Rubio M.A. Ghazvini J. Watanabe K. Simpson L. Suzuki T. EMBO J. 2003; 22: 657-667Crossref PubMed Scopus (94) Google Scholar) (supplemental Fig. S1). E. coli tRNALeu(UUR) having an unmodified wobble uridine was transcribed and purified as described (31Kurata S. Ohtsuki T. Suzuki T. Watanabe K. Nucleic Acids Res. 2003; 31: e145Crossref PubMed Scopus (17) Google Scholar). Ribosomal A-site tRNA Binding—The A-site tRNA binding assay was carried out according to previously described methods (2Ogle J.M. Murphy F.V. Tarry M.J. Ramakrishnan V. Cell. 2002; 111: 721-732Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 27Kirino 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 (211) Google Scholar, 38Ashraf S.S. Sochacka E. Cain R. Guenther R. Malkiewicz A. Agris P.F. RNA. 1999; 5: 188-194Crossref PubMed Scopus (124) Google Scholar) with slight modifications. Briefly, the 5′-ends of E. coli tRNAsLeu(UUR) with or without wobble modifications were labeled with [32P]phosphate and mixed with unlabeled tRNA to adjust the concentration of each tRNA. mRNAs containing A-site UUR codons were synthesized by in vitro run-off transcription using T7 RNA polymerase as described (27Kirino 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 (211) Google Scholar). E. coli 70 S ribosomes were prepared as described (39Spedding G. Ribosomes and Protein Synthesis: A Practical Approach. Oxford University Press, New York1990: 1-30Google Scholar). E. coli tRNAfMet was kindly provided by Dr. Nono Takeuchi (University of Tokyo). The ribosomal P-site was first occupied with initiator tRNAfMet in a mixture (10 μl) consisting of 5.2 pmol of E. coli 70 S ribosome, 2 μg of mRNA, 12.4 pmol of E. coli initiator tRNAfMet, 50 mm Tris-HCl (pH 7.5), 6.5 mm MgCl2, 60 mm KCl, 1 mm DTT, and 2 mm spermine, which was incubated at 37 °C for 17 min. Four different amounts (0.25, 0.5, 0.75, and 1 pmol) of 5′-32P-labeled E. coli tRNAsLeu(UUR) with or without wobble modifications in a mixture (10 μl) consisting of 50 mm Tris-HCl (pH 7.5), 6.5 mm MgCl2, 60 mm KCl, 1 mm DTT, and 2 mm spermine were added to the ribosomal mixtures, and a nonenzymatic binding reaction was performed at 37 °C for 12 min. The reaction mixture was passed through nitrocellulose filters (pore size 0.45 μm; ADVANTEC). The filter was washed with 5 ml of ice-cold buffer consisting of 50 mm Tris-HCl (pH 7.5), 6.5 mm MgCl2, 60 mm KCl, and 1 mm DTT and then air-dried. The amount of tRNA bound was measured by liquid scintillation counting. In Vitro Cell-free Translation—The in vitro cell-free translation assay was carried out according to previously described methods. Briefly, tRNAsLeu(UUR) with or without wobble modifications were leucylated at 37 °C for 10 min in a reaction mixture (30 μl) consisting of 100 mm Tris-HCl (pH 7.6), 5 mm MgCl2, 2 mm ATP, 20 mm KCl, 1 mm DTT, 20% dimethyl sulfoxide, 100 μm [14C]l-leucine, and 1 μg/μl E. coli leucyl-tRNA synthetase. The preparation of E. coli S30 extracts has been described previously (40Takai K. Takaku H. Yokoyama S. Nucleic Acids Res. 1996; 24: 2894-2899Crossref PubMed Scopus (27) Google Scholar). Four UUN-mRNAs, each containing one UUN test codon, were synthesized in vitro using T7 RNA polymerase, as described (41Milligan J.F. Uhlenbeck O.C. Methods Enzymol. 1989; 180: 51-62Crossref PubMed Scopus (1020) Google Scholar), to create the following open reading frame sequence (test codon underlined): 5′-AUGAUCAUUAUCAUUAUCAUUAUCAUAAUCAUCUUNGUGGUGGUCGUGGUGUAAUAGUAG-3′, which encodes Met-Ile10-Leu/Phe-Val5. To construct template DNAs for UUN-mRNAs, DNA fragments were synthesized by Klenow reaction using the following primers: 5′-GAAGGAGATATACATATGATCATTATCATTATCATTATCATAATCATCTTNGTGGTGGTCGTGGTGTA-3′ and 5′-GACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTATGCATCTACTATTACACCACGACCACCAC-3′. The template DNAs were then subjected to PCR to obtain the insert fragments for the templates using the following primers: 5′-CCGGGTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGATC-3′ and 5′-AAAAAAAAAACGAGCCTTTCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATG-3′. Resultant DNAs were inserted into the BamHI/EcoRI site of pUC19 (TOYOBO). The nucleotide sequences of the plasmids encoding UUN-mRNAs were confirmed by the dideoxy termination method of sequencing using a 3100 Genetic Analyzer (Applied Biosystems). Each template DNA was prepared from large scale cultures of E. coli JM109 cells, completely digested with EcoRI, and then transcribed with T7 RNA polymerase. In addition, we prepared an mRNA (GGC-mRNA) in which the UUN codon was replaced with a GGC codon, to be used as a negative control. The cell-free translation reaction (18.7 μl) contained 44 mm HEPES-KOH (pH 7.5), 11 mm DTT, 1.8 mm GTP, 8.4 mm phosphoenolpyruvate potassium salt, 1.5 mm ATP, 0.8% (w/v) polyethylene glycol 8000 (Sigma), 0.54 mg/ml folinic acid calcium salt (Sigma), 44 mm ammonium acetate, 6.4 mm spermidine, 6 mm magnesium acetate, 56 mm potassium glutamate, 0.3 mm each of methionine, isoleucine, and valine, 1 μg of one of the mRNAs, 5 pmol of [14C]Leu-tRNALeu(UUR) with or without wobble modification, and one-sixth volume of S30 extract. The mixture was incubated at 37 °C for 15 min, and the radioactivity of amino acids incorporated into the peptide was measured by liquid scintillation counting. Radioactivity of incorporated Leu into GGC-mRNA was subtracted from that of each UUN-mRNA to obtain decoding activity data. Construction of E. coli ΔmnmE Strain—The E. coli K-12 strain BW25113 (lacIq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBA-DAH33 ΔrhaBADLD78) was used for the "one-step inactivation of chromosomal genes" procedure (42Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11205) Google Scholar). Briefly, a PCR fragment containing the kanamaycin resistance (kan) gene flanked by two flippase recombinase (FLP) recognition targets was generated using pKD4 as the template with the following primers: KO-F, 5′-TAAGCACCGCGCATCCGCCACACAAAGCAACAGGAACATCGTGTAGGCTGGAGCTGCTTC-3′; KO-R, 5′-AGCCGCATCTGACAGTCAGAATGCGGCTTCGTAAGCGCGGCATATGAATATCCTCCTTAGT-3′. This PCR product contains 40 nucleotide extensions that are homologous to the 3′ and 5′ 40 base pairs of mnmE. The PCR fragment was introduced into BW25113/pKD46 E. coli, resulting in insertion of the kan FLP recognition target cassette into the mnmE gene. Disruption of the mnmE gene was confirmed by PCR using 5′-TACATGCTGATGGGTTCCGT-3′ and 5′-GAGGTCACACATATATGTAA-3′ as the primers. The mnmE disruption was transduced into BW25113, and the resistance gene was removed using an FLP expression plasmid (pCP20). Construction of fusA Reporters—The E. coli fusA gene encoding EF-G was cloned into the BamHI/SalI site of pQE-80L (Qiagen). The codons in the fusA gene corresponding to Val-447, Trp-448, and Thr-449 were mutated to AGR codons using a QuikChange mutagenesis kit (Stratagene) according to the manufacturer's instruction to create the fusA reporters using the following primers: 5′-AAGACCCGTCTTTCCGTAGAAGAAGAGACGAAGAATCTAACCAG-3′ and 5′-CTGGTTAGATTCTTCGTCTCTTCTTCTACGGAAAGACGGGTCTT-3′ for fusA-AGA; 5′-ACCCGTCTTTCCGTGTAAGGAGGGACGAAGAATCTAACCAG-3′ and 5′-CTGGTTAGATTCTTCGTCCCTCCTTACACGGAAAGACGGGT-3′ for fusA-AGG. Construction of the reporters was confirmed by DNA sequencing using a 3100 Genetic Analyzer (Applied Biosystems). The pArgU plasmid, a pMW218 harboring the E. coli argU gene, was kindly provided by Dr. Yamada (Mitsubishi Chemical Corp.). Pulse-labeling of Nascent Peptide Chains—Pulse labeling with [35S]methionine was performed as described previously (43Varenne S. Knibiehler M. Cavard D. Morlon J. Lazdunski C. J. Mol. Biol. 1982; 159: 57-70Crossref PubMed Scopus (42) Google Scholar) with slight modifications. Each fusA reporter (fusA-AGA and fusA-AGG) was introduced into an E. coli wild-type or ΔmnmE strain. The cells were grown at 37 °C to OD 0.4 at A600 in LB medium (2 ml), and isopropyl 1-thio-β-d-galactopyranoside was then added to the medium to a final concentration of 1 mm for induction. After a 30-min induction, [35S]methionine (final concentration, 110 μCi/ml) was added to start the pulse labeling (t = 0). At 20 s, unlabeled methionine (final concentration, 20 mm) was rapidly mixed into the medium. Samples (100-μl aliquots) were taken at t = 10, 20, 30, 40, 60, 80, 110, 150, and 200 s and transferred into new tubes containing liquid nitrogen. Before thawing, chloramphenicol was added to a final concentration of 200 μg/ml. The cells were washed twice at 4 °C with double-distilled H2O. Washed pellets were suspended in a mixture (6.5 μl) consisting of 50 mm HEPES-KOH (pH 7.6), 100 mm KCl, 10 mm MgCl2, and 0.2 mm phenylmethylsulfonyl fluoride. To this suspension, 2.5 μl of sample buffer (250 mm Tris-HCl (pH 6.8), 40% glycerol, 8% SDS, and 0.005% bromophenol blue) and 1 μl of 2-mercaptoethanol were added, and the samples were then boiled at 95 °C for 5 min. The lysates were analyzed by SDS-PAGE on a 10–20% polyacrylamide gradient gel (Wako Chemicals). The gel was stained with Coomassie Brilliant Blue R250, washed, and vacuum-dried. The gel was exposed to an imaging plate, and the labeled polypeptides were visualized using a bioimaging analyzer (BAS 5000; Fuji Photo Film). Northern Blots—Total RNA from E. coli was isolated using ISOGEN (Nippon Gene), according to the manufacturer's instructions. The total RNA (∼5 μg) was electropho
Referência(s)