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tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli

2002; Springer Nature; Volume: 21; Issue: 14 Linguagem: Inglês

10.1093/emboj/cdf362

1460-2075

Jeannette Wolf, André P. Gerber, Walter Keller,

RNA and protein synthesis mechanisms

Article15 July 2002free access tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli Jeannette Wolf Jeannette Wolf Department of Cell Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland Search for more papers by this author André P. Gerber André P. Gerber Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305-5307 USA Search for more papers by this author Walter Keller Corresponding Author Walter Keller Department of Cell Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland Search for more papers by this author Jeannette Wolf Jeannette Wolf Department of Cell Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland Search for more papers by this author André P. Gerber André P. Gerber Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305-5307 USA Search for more papers by this author Walter Keller Corresponding Author Walter Keller Department of Cell Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland Search for more papers by this author Author Information Jeannette Wolf1, André P. Gerber2 and Walter Keller 1 1Department of Cell Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland 2Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305-5307 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3841-3851https://doi.org/10.1093/emboj/cdf362 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We report the characterization of tadA, the first prokaryotic RNA editing enzyme to be identified. Escherichia coli tadA displays sequence similarity to the yeast tRNA deaminase subunit Tad2p. Recom binant tadA protein forms homodimers and is sufficient for site-specific inosine formation at the wobble position (position 34) of tRNAArg2, the only tRNA having this modification in prokaryotes. With the exception of yeast tRNAArg, no other eukaryotic tRNA substrates were found to be modified by tadA. How ever, an artificial yeast tRNAAsp, which carries the anticodon loop of yeast tRNAArg, is bound and modified by tadA. Moreover, a tRNAArg2 minisubstrate containing the anticodon stem and loop is sufficient for specific deamination by tadA. We show that nucleotides at positions 33–36 are sufficient for inosine formation in mutant Arg2 minisubstrates. The anticodon is thus a major determinant for tadA substrate specificity. Finally, we show that tadA is an essential gene in E.coli, underscoring the critical function of inosine at the wobble position in prokaryotes. Introduction The nucleotide inosine (I) has been observed in viral transcripts and eukaryotic mRNAs. In all known cases, I results from the deamination of adenosine (A), a process termed RNA editing. Because I is read as guanosine (G) by the translational machinery (Basilio et al., 1962), RNA editing can change codon specificity and therefore the amino acid sequence of the encoded protein, resulting in multiple protein products with different biological function from a single mRNA precursor. In mammals, for example, the mRNA precursors (pre-mRNAs) coding for subunits of glutamate-gated ion channel receptors (GluRs) and the serotonin receptor subunit 5-HT2C are edited (reviewed in Seeburg et al., 1998; Gott and Emeson, 2000; Maas and Rich, 2000; Gerber and Keller, 2001; Keegan et al., 2001). Editing was also detected in Drosophila melanogaster, Caenorhabditis elegans and in hepatitis delta virus (Polson et al., 1996; Smith et al., 1996; Morse and Bass, 1999; Semenov and Pak, 1999; Hanrahan et al., 2000). RNA editing therefore represents an important mechanism for increasing the genetic diversity in eukaryotes. In all these cases, pre-mRNA editing requires double-stranded RNA (dsRNA) structures, which are formed between exonic sequences encompassing the editing site and downstream intronic sequences. RNA editing of pre-mRNAs is catalyzed by adenosine deaminases acting on RNA (ADARs; Bass et al., 1997). ADARs have a common modular organization consisting of two or three dsRNA-binding domains (dsRBD) and a catalytic deaminase domain containing three Zn2+-chelating residues and a proton-shuffling glutamate (reviewed in Bass, 1997; Maas and Rich, 2000; Gerber and Keller, 2001). Inosine is not only present in mRNAs, but also in tRNAs. It was first found in tRNAAla from yeast (Holley et al., 1965). Eukaryotic tRNAAla contains I at two positions: at the wobble position of the anticodon (position 34) and as the derivative N1-methylinosine (m1I) at position 37, 3′ adjacent to the anticodon (Holley et al., 1965; reviewed in Grosjean et al., 1996). In eukaryotes, seven to eight tRNAs contain I at position 34, whereas in prokaryotes and plant chloroplasts only tRNAArg2 contains this modification. As in pre-mRNAs, I in tRNAs is the product of hydrolytic deamination of genomically encoded A (Auxilien et al., 1996). m1I at position 37 is formed in a two-step reaction. First, A is deaminated to I, which is further methylated by a methyltransferase (Grosjean et al., 1996; Björk et al., 2001). The genomes of Saccharomyces cerevisiae and prokaryotes do not encode classical ADAR proteins, but, based on sequence homology to ADARs, a yeast protein was identified that contains a deaminase domain but lacks a known RNA-binding motif. This protein catalyzes the deamination of A at position 37 in yeast tRNAAla and was therefore named adenosine deaminase acting on tRNA 1 and its gene tRNA-specific adenosine deaminase 1 (scADAT1/TAD1; Gerber et al., 1998). Tad1p specifically targets A37 in tRNAAla. Mutations affecting the three-dimensional structure of the tRNA or the length of the anticodon loop abolish conversion of A to I in vitro (Gerber et al., 1998). Whereas A37 is unmodified in a Δtad1 strain, modification of A34 was unaffected in all tRNAs tested, suggesting that I formation at these two positions is catalyzed by different enzymes. Whereas I in the wobble position (I34) is crucial to allow the decoding of three codons by a single tRNA (Crick, 1966), the function of m1I37 is less clear. It was postulated that this modification may prevent translational frameshifts and improve translational fidelity (Björk et al., 1989). ADAT1 proteins have also been cloned from human (Maas et al., 1999), mouse (Maas et al., 2000) and D.melanogaster (Keegan et al., 2000). The tRNA adenosine deaminase that specifically deaminates A34 has been partially purified from yeast extracts (Auxilien et al., 1996) and has been identified by homology to Tad1p (Gerber and Keller, 1999). Activity depended on the correct tRNA structure as well as on the length and the nucleotide sequence of the anticodon loop. The tRNA:A34 deaminase is a heterodimer of sequence-related subunits named scADAT2/Tad2p and scADAT3/Tad3p. Both polypeptides contain a deaminase domain that resembles that of the cytidine deaminase (CDA) superfamily (Gerber and Keller, 1999), although Tad2p/Tad3p deaminates A. Most cytidine/deoxycytidylate deaminases catalyze deaminations of mononucleotides, but there are also a few known cytidine deaminases that act on RNA (CDARs). One of the best studied examples is APOBEC1, which catalyzes the deamination of a cytidine to uridine in the apoB mRNA, resulting in a change of a glutamine codon to a stop codon (reviewed by Chester et al., 2000). Interestingly, the deaminase domain of Tad3p lacks the glutamate that, analogous to CDAs and CDARs, is thought to be part of the active center. The lack of this glutamate suggests that Tad3p is catalytically inactive and that Tad2p is the catalytic subunit of the complex. Deamination of tRNA is not limited to eukaryotes, but has also been detected in Escherichia coli extracts (Auxilien et al., 1996). In E.coli, the only known A to I conversion in RNA is the deamination of tRNAArg2 at position 34. Based on sequence homology, we identified an E.coli protein homologous to Tad2p. Here, we report the identification and characterization of this enzyme, which is necessary and sufficient to catalyze this editing reaction. tadA/ecADAT2 shows sequence homology to yeast Tad2p and Tad3p and is encoded by an essential gene, thus underlining the vital function of I34. The identification of the first prokaryotic tRNA-specific adenosine deaminase also provides further insight into the evolution of the deaminase family of enzymes. Results tadA acts specifically on tRNAArg2 Database searches with the S.cerevisiae Tad2p sequence and the FASTA program (Pearson and Lipman, 1988) revealed that the E.coli open reading frame (ORF) yfhC is 34% identical to Tad2p (Figure 1). To determine whether yfhC indeed encodes an adenosine deaminase, the protein was overexpressed in E.coli and purified to apparent homogeneity (Materials and methods; Figure 2A and B). Figure 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). Download figure Download PowerPoint Figure 2.tadA specifically deaminates A34 in tRNAArg2. 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 tRNAArg2 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 '–', tRNAArg2 was incubated with buffer only. (D) Sequence analysis of in vitro modified tRNAArg2. 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: tRNAArg2 incubated with buffer only. Download figure Download PowerPoint Peak fractions of the final gel filtration column were tested for adenosine deaminase activity on in vitro transcribed and uniformally [α-33P]ATP-labeled tRNAArg2. The recombinant protein indeed converted A to I (Figure 2C). This activity stemmed from the recombinant protein and not from an endogenous enzyme, since glutathione S-transferase (GST) purified from E.coli or eluates from Ni2+–agarose incubated with extract from empty-vector-transformed cells had no activity (results not shown). A 2 ng aliquot of recombinant yfhC protein formed up to 0.5 mol I/mol tRNA (Figure 2C, fraction 12) and no other protein was needed for activity (Figure 2C, fraction 12). This is in contrast to the yeast enzyme, which acts as a heterodimer composed of Tad2p and Tad3p (Gerber and Keller, 1999). To determine the site in tRNAArg2 that is modified by recombinant yfhC, in vitro modified tRNA was amplified by RT–PCR and sequenced. Since I base-pairs with C during reverse transcription, A to I deamination changes the sequence from A to G. tRNAArg2 incubated with recombinant yfhC contained a G at position 34, demonstrating that the template-encoded A at this position was deaminated to I. Furthermore, sequencing of tRNAArg2 revealed that ∼90% of A34 was deaminated to I, as can be seen from the ratio of G to A at position 34 (Figure 2D). tRNAArg2 incubated in the absence of protein carried A at position 34 and therefore was not modified (Figure 2D). No other A in tRNAArg2 was modified by the recombinant protein. These results showed that the yfhC gene encodes a protein that is sufficient to reconstitute A34:tRNA deaminase activity in vitro. Therefore, we renamed yfhC as tadA (for tRNA adenosine deaminase A). The specificity of recombinant tadA was tested by comparing its activity on various tRNA substrates (Figure 3A). In E.coli, only one tRNA substrate is known that is deaminated at position 34 (tRNAArg2), in contrast to S.cerevisiae where seven different tRNAs are substrates for Tad2p/Tad3p (Auxilien et al., 1996; Gerber and Keller, 1999). Auxilien et al. (1996) showed that not only tRNAArg2, but also a variant of tRNAAsp from S.cerevisiae carrying the anticodon loop of yeast tRNAArg, are deaminated in vitro upon incubation with extracts from E.coli. The latter observation could be reproduced with recombinant tadA, which forms up to 0.8 mol of I in this artificial tRNA, suggesting that the anticodon loop carries important recognition signals for the enzyme (Figure 3A, lane 4). This hypothesis is supported by the result that tadA deaminated yeast tRNAArg with the same efficiency as E.coli tRNAArg2 (Figure 3A, lanes 10 and 11). These two tRNAs have a conserved anticodon loop sequence (Figure 3B). However, parts of the anticodon stem and the acceptor stem are also conserved between yeast and E.coli tRNAArg, and these nucleotides might also contribute to the recognition of the tRNA by tadA (Figure 3B). Figure 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 tRNAAla from S.cerevisiae (lanes 1 and 2), a tRNAAsp mutant from S.cerevisiae (lanes 3 and 4), tRNAAla from B.mori (lanes 5 and 6), human tRNAAla (lanes 7 and 8), S.cerevisiae tRNAArg (lanes 9 and 10) and tRNAArg2 from E.coli (lane 11) were used. Mock incubation with WT yeast tRNAAsp 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 tRNAArg2, S.cerevisiae tRNAArg and S.cerevisiae tRNAAsp with the anticodon loop of tRNAArg. 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, tRNAArg2; hAla, human tRNAAla; B.m. Ala, B.mori tRNAAla; S.c. Ala, S.cerevisiae tRNAAla. Download figure Download PowerPoint tRNAAla from S.cerevisiae, Bombyx mori and Homo sapiens are not deaminated by tadA, although they are known to contain I34 in vivo (Figure 3A, lanes 2, 6 and 8). Notably, all these tRNAs are substrates for yeast Tad2p/Tad3p (Figure 3A, lanes 1, 5 and 7; Auxilien et al., 1996; Gerber and Keller, 1999). In yeast and B.mori, tRNAAla positions 34 and 37 are deaminated and 2 mol I/mol tRNA are generated upon incubation with yeast extract (Figure 3A, lanes 1 and 5). The substrate specificity of tadA could also be shown with UV-cross-linking experiments that indicate binding of the protein to the tRNA substrate. Recombinant GST–tadA could be UV cross-linked to its natural substrate tRNAArg2 (Figure 3C, lane 2), but not to tRNAAla from H.sapiens, B.mori or S.cerevisiae (Figure 3C, lanes 4–6). GST alone could not be cross-linked to any of these substrates (results not shown). In summary, we showed that recombinant tadA specifically deaminates tRNAArg2 at position 34. To determine whether the anticodon stem and loop is sufficient for deamination by tadA, a tRNAArg2 minisubstrate containing only the anticodon stem and loop was tested in vitro (Figure 4A). This minisubstrate was deaminated as efficiently as full-length tRNAArg2 (Figure 4B, lane 1; Table I). In the majority of the experiments, deamination of wild-type (WT) Arg2 minisubstrate yielded between 1.2 and 1.4 mol I/mol tRNA, which is slightly higher than expected. Interestingly, when A at the wobble position was mutated to G, the minisubstrate was not deaminated, indicating that this nucleotide is essential for tadA recognition (Figure 4B, lane 2). It is possible that a second A was deaminated in a fraction of the WT minisubstrate. However, we consider this possibility unlikely because no I was formed when A34 was mutated and deamination of the second A would thus depend on the presence of A34. Although the anticodon arm of Arg2 serves as a substrate for tadA, there seem to be differences in site specificity between the full-length tRNAArg2 and mini Arg2. Sequences and structures outside the anticodon arm might be important for deamination at the correct position. In order to determine nucleotides that are essential to specify these short RNAs as substrates for tadA, several mutant minisubstrates were generated and tested in vitro. No I was formed with minisubstrates that have mutations at position 35 or 36 in the anticodon (Figure 4B, lanes 3 and 4). This showed that nucleotides in the anticodon were key determinants for tadA activity. The importance of the anticodon loop size was investigated with a minisubstrate that has an 8 nucleotide (nt) loop. This substrate was still deaminated, but with a lower efficiency compared with the WT RNA (Figure 4B, lane 5; Table I), whereas a 3 nt loop was not sufficient for tadA activity (Figure 4B, lane 6). A minisubstrate that has a random loop sequence but the correct anticodon was not deaminated, showing that the anticodon is not the only determinant for tadA activity (Figure 4B, lane 7). When position 37 in mutant 6 was changed to G, I formation was not restored (Figure 4B, lane 8). G at position 37 in the WT Arg2 minisubstrate did not affect tadA activity (results not shown). However, reverting position 33 in mutant 6 to the WT U residue restored I formation, which was, however, not as efficient as with WT minisubstrate (Figure 4B, lane 9; Table I). This suggests that U33 is an important determinant for tadA activity, but additional nucleotides probably contribute to efficient deamination. To determine whether a stem–loop structure in addition to the sequence is required for tadA activity, a linear minisubstrate was analyzed (mutant 9, Figure 4A). Although mutant 9 contains the same essential nucleotides for activity as mutant 7, the linear RNA was not deaminated (Figure 4B, lane 10), indicating that a stem–loop structure is essential for tadA activity. As expected, the anticodon arm of E.coli tRNAArg3 was not a substrate for tadA (Figure 4B, lane 11) because this RNA does not contain A at the wobble position (Figure 4A). However, I formation was detected in Arg3 when C34 was mutated to A (Figure 4B, lane 12). This result provides further information about key nucleotides for tadA activity. The anticodon stem of Arg3 differs from that of Arg2, indicating that recognition of the stem by tadA is not sequence specific (Figure 4A). In summary, a minisubstrate with a stem–loop structure is deaminated at the wobble position of the anticodon if the RNA has the sequence UACG within the loop. However, it cannot be excluded that other regions of the tRNA are needed for efficient deamination. The results with the mutant minisubstrates suggest that, in addition to the correct sequence in the loop, the structure and the loop size, but not the stem sequence, are important for deamination by tadA. Figure 4.RNA minisubstrates derived from E.coli tRNAArg2 and tRNAArg3 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). Download figure Download PowerPoint Table 1. Quantification of tadA activity obtained with tRNA-derived minisubstrate RNAs Substrate Mutations compared with WT mol I/mol RNA Arg2 WT 1.2–1.4 mutant 1 A34G 0 mutant 2 C35G 0 mutant 3 G36C 0 mutant 4 8 nt loop, U28G, A42C 0.5 mutant 5 3 nt loop, 7 bp G-C stem 0 mutant 6 U28G, C32U, U33C, A37C, A38U, A42C 0 mutant 7 U28G, C32U, U33C, A37G, A38U, A42C 0 mutant 8 U28G, C32U, A37C, A38U, A42C 0.26 mutant 9 Linear 0 Arg3 WT 0 mutant 10 C34A 1.39 A point mutation in tadA leads to an inactive enzyme in vitro Next we asked whether tadA is responsible for I formation at position 34 of tRNAArg2 in vivo and took advantage of a non-lethal point mutant described previously (Poulsen et al., 1992). The mutant form of tadA carries a glutamic acid instead of an aspartic acid at position 64 (D64E; Figure 1). This amino acid is highly conserved in bacterial, but not in eukaryotic, Tad2 proteins. To determine whether deamination of A34 in tRNAArg2 is affected by this mutation in vivo, total RNA was isolated from the mutant (NWL37) and the corresponding parental strain (NWL32). After amplification of tRNAArg2 by RT–PCR with specific primers, the cDNAs were subcloned and multiple clones of each strain were sequenced. As expected, all clones derived from the WT strain contained G at position 34 (results not shown). Interestingly, 16 out of 17 clones derived from NWL37 also had G at position 34, and only one clone contained A at this position (results not shown). Therefore, this point mutant of tadA is almost fully active in vivo, which is not suprising given that the mutant strain showed no growth defect even at 42°C (results not shown), even though tadA is an essential gene. Although the mutant protein was functional in cells, its activity might be reduced in an in vitro assay system. We therefore prepared extracts from mutant and parental strains, and tested equal amounts in a deamination assay. Whereas extracts from WT cells showed activity (NWL32; Figure 5, lane 2), no I formation could be detected with extracts from the mutant strain (NWL37; Figure 5, lane 3). However, activity could be restored by transforming the mutant cells with a tadA expression construct (NWL37R; Figure 5, lane 4). Thus, the mutation D64E abolishes the enzymatic activity of tadA in vitro, although it does not detectably affect its activity in cells. Figure 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). Download figure Download PowerPoint tadA cannot substitute for yeast Tad2p in vivo and in vitro Because tadA is 34% identical to yeast Tad2p and is functionally conserved, we attempted to replace Tad2p with tadA in a ΔTAD2 yeast strain (Gerber and Keller, 1999). tadA was cloned into the pGALΔTrp-FLIS6 vector (Gerber et al., 1998) and transformed into a Δtad2 yeast strain carrying a copy of TAD2 on a plasmid with the URA3 marker (pFL38; Gerber and Keller, 1999). Cells were then transferred to plates containing 5-fluoro-orotic acid (5-FOA) and galactose to force the loss of the URA3-marked TAD2 plasmid and induce the expression of tadA, respectively. No cells grew, showing that tadA cannot substitute for Tad2p in vivo. Next we tested whether tadA could functionally replace Tad2p in an in vitro deaminase assay. No activity was detected when tadA pre-incubated with yeast Tad3p was incubated with B.mori tRNAAla, which is a substrate for the Tad2p/Tad3p heterodimer (Figure 6A, lanes 4 and 2). Thus, tadA cannot replace yeast Tad2p either in vivo or in vitro. Figure 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 tRNAAla 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. Download figure Download PowerPoint tadA forms homodimers Because yeast Tad2p functions as a heterodimer with Tad3p, and E.coli does not encode a Tad3-like protein, we wondered whether tadA might form homodimers instead. This hypothesis was tested with three different approaches. We investigated the interaction of Flag-tadA and in vitro translated [35S]methionine-labeled tadA in a Flag pull-down assay. The tagged tadA and labeled translated tadA were pre-incubated, bound to anti-Flag–agarose, washed and eluted by boiling the matrix in SDS–gel loading buffer. The result is shown in Figure 6B (lane 2). Approximately 10% of the tadA input was pulled down by Flag-tadA. In a second experiment, recombinant Flag-tadA was chromatographed on a gel filtration column, which separates proteins according to size, and fractions were tested for tadA activity. Fifty percent of tadA was eluted at a position corresponding to a dimer and ∼50% of tadA was eluted as a monomer (results not shown). This result suggests that tadA may act as a homodimer. However, the presence of tadA monomers indicates that tadA does not interact as strongly with itself as does yeast Tad2p with Tad3p (Gerber and Keller, 1999). Formation of tadA monomers could occur by separation of tadA dimers on the gel filtration column due to the experimental conditions. To confirm dimerization, we carried out chemical cross-linking experiments and, indeed, observed a complex corresponding in size to tadA homodimers (Figure 6C, lanes 2–4). Complex formation was strongly stimulated by the addition of BSA, although a weak cross-link was also detectable in some experiments in the absence of BSA (results not shown). The reason for this stimulation is unclear; however, BSA might have a stabilizing effect on the interaction of the highly purified tadA subunits. tadA complex formation was specific and only involved tadA subunits but not BSA. Complexes with BSA would have a higher molecular mass and could therefore easily be distinguished from tadA homodimers by size. Dimeriza tion was not complete and could be observed for ∼30% of tadA. This might be due to the experimental procedure in that tadA might have been cross-linked under suboptimal dimerization conditions. The addition of E.coli total tRNA did not influence dimer formation in these assays, indicating that the observed dimerization did not require substrate tRNA (results not shown). These experiments indicate that tadA can form a homodimer in vitro; however, it remains to be shown whether it also acts as a homodimer in vivo. tadA is essential for cell viability tadA has previously been reported to be a non-essential gene in E.coli (Poulsen et al., 1992). tadA was deleted by inserting a chloramphenicol acetyltransferase (CAT) gene into the SphI restriction site of the ORF, which removed the last 17 amino acids of the protein. We re-investigated whether tadA is essential for viability because recombinant tadAΔC, corresponding to the knock-out gene described by Poulsen et al. (1992), was active in a tRNA editing assay (results not shown). The tadA gene was inactivated with the two-step sacB counterselection technique (Reece and Phillips, 1995). For this purpose, a plasmid-borne tadA gene was produced, which was disrupted by an interposon carrying a kanamycin-resistance cassette (pJW30; Figure 7A). The disrupted tadA