Fig. 5. The point mutation D64E abolishes tadA activity in vitro. Lane 1, no protein; lanes 2–4, 40 µg of total protein of E.coli extract. All reactions were incubated for 2 h at 37°C and processed as described in the legend to Figure 2 (see also Materials and methods).

Fig. 1. Bacterial genomes encode a protein (tadA) related to Tad2p of S.cerevisiae. Multiple sequence alignment of E.coli tadA, yeast Tad2p and putative tadA sequences from different bacteria. Residues conserved in >83% of the proteins are shown in black, similar amino acids in gray. The three putative Zn²⁺-chelating residues (diamond) and the glutamate thought to mediate proton transfer (bullet) are marked. The position of the point mutation (D64E) in the tadA mutant NWL37 is indicated by an asterisk. The alignment was generated with the Clustal_W software at the European Bioinformatics Institute (Thompson et al., 1994).

Fig. 4. RNA minisubstrates derived from E.coli tRNA^Arg2 and tRNA^Arg3 are sufficient for deamination by tadA in vitro. (A) Schematic drawing of Arg minisubstrates that were tested in vitro. Nucleotides shown in gray are mutated compared with WT Arg2. A at the wobble position of the anticodon is shown in bold. Mutations in each minisubstrate are indicated by arrows. (B) Editing assay with minisubstrates and tadA. A 25–100 fmol aliquot of minisubstrate, 100 ng of recombinant Flag-tadA-His₆ and 500 ng of BSA were incubated in a total volume of 25 µl. WT Arg2 (lane 1), mutant 1 (lane 2), mutant 2 (lane 3), mutant 3 (lane 4), mutant 4 (lane 5), mutant 5 (lane 6), mutant 6 (lane 7), mutant 7 (lane 8), mutant 8 (lane 9), mutant 9 (lane 10), WT Arg3 (lane 11) and mutant 10 (lane 12) were used. All reactions were incubated for 45 min at room temperature and processed as described in the legend to Figure 2 (see also Materials and methods).

Fig. 6. tadA cannot replace yeast Tad2p in vitro and forms homodimers. (A) Recombinant yeast Tad2p, Tad3p and E.coli tadA were pre-incubated as indicated at the top and tested for activity in vitro with tRNA^Ala from B.mori. As a control, the yeast Tad2p/Tad3p complex was used (lane 2). (B) Flag pull-down assay with in vitro translated tadA. ³⁵S-labeled tadA was incubated with Flag-tagged tadA (lane 2) or buffer (lane 1) and subsequently bound to Flag–agarose and eluted. Ten percent of the input is shown in lane 3. Proteins were separated on a 12% SDS–polyacrylamide gel. Molecular masses of the protein standards are indicated in kDa on the left. (C) Chemical cross-linking of recombinant tadA. A 300 ng aliquot of tadA (lanes 2–4) was incubated in a reaction mixture containing tris(2,2′-bipyridyl) dichlororuthenium(II)hexahydrate, APS and BSA (90 ng, 0.9 µg and 9 µg, respectively). tadA incubated with buffer only is shown in lane 1. Reaction products were separated on a 12% SDS–polyacrylamide gel and blotted onto a nitrocellulose membrane. tadA was detected with a mouse anti-Flag antibody. Molecular masses of protein standards are indicated in kDa on the left.

Fig. 7. tadA is an essential gene. (A) The sacB counterselection procedure. The wild-type chromosomal copy of tadA was exchanged via a two-step homologous recombination reaction with a gene copy disrupted by the insertion of a kanamycin-resistance cassette. After the second recombination step, colonies were analyzed by their antibiotic-resistance profile for the presence or absence of the Kan cassette in the tadA locus and for mutational inactivation of the sacB gene. The mutation in sacB is indicated by an asterisk. (B) Summary of the clones obtained by the strategy described in (A). The total number of sucrose-resistant colonies analyzed is indicated in the first column (Total clones). The number of isolated colonies with a disrupted tadA (ΔtadA) gene in the chromosome is shown in the second column and the number of colonies with a restored WT tadA locus (WT) after the second recombination step is shown in the third column. Colonies that had acquired sucrose resistance through sacB inactivation are listed in the fourth column (sacB^–). Plasmids pJW30 and pJW117 carry disrupted and WT copies of the tadA ORF, respectively. (C) PCR analysis of WT (lane 1) and ΔtadA (lane 2) genomic E.coli DNA. Primers are indicated by arrows and anneal 500 bp upstream of the tadA ORF and at the 3′ end of the ORF, respectively. Sizes of standards (lane M) are indicated in bp on the left.

Fig. 2. tadA specifically deaminates A₃₄ in tRNA^Arg2. SDS– polyacrylamide gel stained with silver (A), western blot (B) and tRNA editing assay (C) of the final gel filtration column of the purification of recombinant tadA. A 20 µl aliquot of each fraction was separated on a 12% SDS–polyacrylamide gel (A) and transferred to a nitrocellulose membrane for detection with antibodies (B). The western blot was probed with a mouse α-GST monoclonal antibody. Fraction numbers are indicated at the top, the molecular masses of the size standards in kDa on the left. (C) Two microliters of the fractions indicated at the top were incubated with [³³P]ATP-labeled tRNA^Arg2 for 30 min at 37°C. Reactions were treated with P1 nuclease and the products separated by one-dimensional TLC. The positions of AMP, IMP and the origin are indicated on the right. The position of IMP was verified with unlabeled 5′ IMP. In lane ‘–’, tRNA^Arg2 was incubated with buffer only. (D) Sequence analysis of in vitro modified tRNA^Arg2. The nucleotide sequence surrounding the anticodon is shown. tRNA from reactions shown in (C) was amplified by RT–PCR and sequenced. The nucleotide at position 34 is shaded gray, the anticodon is underlined. Control: tRNA^Arg2 incubated with buffer only.

Fig. 3. Substrate specificity of tadA. (A) tRNA-editing assay with tadA and different tRNAs. tRNA substrates were incubated with either 2 ng of recombinant His₆-tadA (lanes 2, 4, 6, 8, 10 and 11), 20 ng of recombinant scTad2p/scTad3p (lane 7) or 40 µg of S.cerevisiae total protein (lanes 1, 3 and 5). One hundred femtomoles of tRNA^Ala from S.cerevisiae (lanes 1 and 2), a tRNA^Asp mutant from S.cerevisiae (lanes 3 and 4), tRNA^Ala from B.mori (lanes 5 and 6), human tRNA^Ala (lanes 7 and 8), S.cerevisiae tRNA^Arg (lanes 9 and 10) and tRNA^Arg2 from E.coli (lane 11) were used. Mock incubation with WT yeast tRNA^Asp did not result in I formation (result not shown). All reactions were incubated for 1 h at 30°C and processed as described in the legend to Figure 2 (see also Materials and methods). (B) Cloverleaf structure of E.coli tRNA^Arg2, S.cerevisiae tRNA^Arg and S.cerevisiae tRNA^Asp with the anticodon loop of tRNA^Arg. Completely conserved nucleotides and nucleotides conserved as purines or pyrimidines between tRNAs from different species are shown in red (Klingler and Brutlag, 1993). Nucleotides that are conserved in addition between the three tRNAs are depicted in blue. (C) UV-cross-linking experiments of recombinant tadA and different tRNAs. A 400 ng aliquot of GST–tadA (lanes 2–5) and 100 fmol of [³³P]ATP-labeled tRNA were irradiated and samples treated with RNaseA. Proteins were separated on a 12% SDS–polyacrylamide gel and gels exposed to a phosphoimager screen. E.c., E.coli; S.c., S.cerevisiae; B.m., B.mori; Arg2, tRNA^Arg2; hAla, human tRNA^Ala; B.m. Ala, B.mori tRNA^Ala; S.c. Ala, S.cerevisiae tRNA^Ala.