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Cells were transformed with PCR-amplified fragments bearing the TRP1 marker gene flanked by TAD2 or TAD3 sequences, respectively. Tryptophan prototrophs were selected and correct genomic integration of the TRP1 gene was verified by PCR. In tad2Δ cells the coding sequence from amino acids 74 to 210 of TAD2 was replaced by the TRP1 marker, and in tad3Δ cells, amino acids 152 to 233 were replaced.
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TAD2 and TAD3 were disrupted in the diploid S. cerevisiae strain BMA41 [N. Amrani, M.-E. Dufour, N. Bonneaud, F. Lacroute, Mol. Gen. Genet. 252, 552 (1996)]. Cells were transformed with PCR-amplified fragments bearing the TRP1 marker gene flanked by TAD2 or TAD3 sequences, respectively. Tryptophan prototrophs were selected and correct genomic integration of the TRP1 gene was verified by PCR. In tad2Δ cells the coding sequence from amino acids 74 to 210 of TAD2 was replaced by the TRP1 marker, and in tad3Δ cells, amino acids 152 to 233 were replaced.
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0345332430
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note
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6-Tad2.
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17
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0344469689
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note
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153 (CTG) are changed to Ser (ACT) and Pro (CCG), respectively.
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19
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0345332428
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note
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6 were passed twice over 400 μl of an anti-M2-FLAG affinity matrix (Kodak) and eluted with buffer B [buffer A containing 25 mM KCl, 0.1 mM EDTA, and 0.1 mM dithiothreitol (DTT) instead of β-mercaptoethanol] supplemented with FLAG-peptide (50 μg/ ml) (Kodak). Fractions containing tagged Tad2p were loaded on a MonoQ column (PC 1.6/5; Smart System, Pharmacia) that had been equilibrated in buffer C (buffer B containing 0.2 mM EDTA and 1 mM DTT) and developed with a 2-ml gradient from 25 to 300 mM KCl. Fifty-microliter fractions were collected. Fractions with tRNA editing activity eluted at 145 mM KCl (∼4 μg).
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0345332427
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note
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2) is not the correct start codon but part of a second intron. To confirm this, we amplified the TAD3 ORF with primers YL8 (5′-GGAC-TAGTTAAGAAAGTTAATAATCCGC-3′) and YL2 (5′-CGACTAGTCCGCAGCAGACATCCCGGTCAAC-3′) on S. cerevisiae cDNA. Spe I sites for subcloning are underlined. TAD3 with introns was obtained by PCR on genomic DNA with primers YLreg1 (5′-GGTCTG-TAGATCAATGTCAAGC-3′) annealing 237 bp upstream and YLreg2 (5′-GTTCAAGCAGCAACTA-CAGTCG-3′) hybridizing 386 bp downstream of the TAD3 ORF. The fragment was further cloned into pFL38 and pFL36, respectively (27).
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0033034668
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0344038528
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note
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2 (15).
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0344469685
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note
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6-Tad3. Plasmids were transformed into BL21 (DE3). Escherichia coli cells and protein expression were induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside for 5 hours at 23°C. Cells were lysed by sonication and the extract was fractionated on a Ni-NTA column and on anti-FLAG-M2 affinity matrix. rTad3p was further purified on a 1-ml HiTrap Q column (Pharmacia).
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0026573580
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with primers YJ-E56A (5′-GTAG-CACATGCTGCGTTCATGGGGATC-3′) to generate pTrc-FLIS6-Tad2E56A and YL-V218E (5′-CATCGAC-CACAGTGAAATGGTGGGATC-3′) for pTrc-FLIS6-Tad3V218E
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Site-directed mutagenesis was carried out as described [I. Mikaelian and A. Sergeant, Nucleic Acids Res. 20, 376 (1992)] with primers YJ-E56A (5′-GTAG-CACATGCTGCGTTCATGGGGATC-3′) to generate pTrc-FLIS6-Tad2E56A and YL-V218E (5′-CATCGAC-CACAGTGAAATGGTGGGATC-3′) for pTrc-FLIS6-Tad3V218E.
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0345332425
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We thank H. Langen (Hoffmann La-Roche) for the mass spectrometry analysis; H. Grosjean, J. Atkins, and H. Himeno for the tRNA templates; and members of our laboratory and M. A. O'Connell for critical reading of the manuscript Supported by Boehringer Ingelheim Fonds (A.P.G.), the University of Basel, the Swiss National Science Foundation, the Human Frontiers Science Program, and the Louis-Jeantet Foundation for Medicine.
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