Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation

Abstract

The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNAGln amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated Glu-tRNAGln, functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNAGln in B. subtilis and that glutamyl-tRNAGln amidotransferase is a novel and essential component of the translational apparatus.

Figure 1

The transamidation pathway of Gln-tRNA^Gln formation

Figure 2

Glu-AdT gene arrangement. The genomic organization of the Glu-AdT operon is shown. This includes −35 and −10 regions of the putative promoter, the Shine-Dalgarno (SD) sequence, and the putative transcription start +1.

Figure 3

Analysis of Glu-AdT fractions during purification. Proteins were separated on SDS/16.5% PAGE in tricine (pH 8.4) of samples from the purification steps. Lanes: A, Fraction I [S100 extract of E. coli BL21(DE3)/pCAB]; B, Fraction II (Q Sepharose); C, Fraction III (gel filtration); D, Fraction IV (Mono Q).

Figure 4

Immunoblot analysis of Glu-AdT expressed from various clones. Lane 1, purified Glu-AdT complex. S100 fractions from lane 2, E. coli BL21(DE3); lane 3, B. subtilis; lane 4, E. coli BL21(DE3)/pCAB; lane 5, E. coli BL21(DE3)/pCA; lane 6, E. coli BL21(DE3)/pA; lane 7, E. coli BL21(DE3)/pB; lane 8, E. coli BL21(DE3)/pC.

Figure 5

Activity of Glu-AdT holoenzyme and subunits. Phosphoroimage of thin-layer chromatography separation of ¹⁴C-labeled Gln and Glu derived from deacylation of aminoacyl-tRNA recovered from the assay (see text). Glu-AdT was from S100 extracts of E. coli BL21(DE3) harboring: Lane 1, pCAB; lane 2, pAB; lane 3, pCA; lane 4, pA; lane 5, pB; lane 6, pC; lane 7, S100 extracts from pCA and pB mixed in vitro; lane 8, S100 extracts from pA, pB, and pC mixed in vitro.

Figure 6

Alignment of gatA, gatB, and gatC genes with related sequences. Alignments were created with the Genetics Computer Group, clustalw, and boxshade programs. The database accession numbers are shown in parentheses. Red represents residues that are completely conserved in at least 85% of the sequences, blue those residues that are functionally conserved within the sequences, and black the other residues. (A) gatC genes. afu1, Archaeoglobus fulgidus (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-tigrbl); bsu1, B. subtilis (AF008553); mca1, M. catarrhalis (U49269); mja1, Methanococcus jannaschii (U67558) and also MJ0243; ngo1, Neisseria gonorrhoeae (http://dna1.chem.uoknor.edu/gono.html); sau1, Staphylococcus aureus (U06451); spy1, Streptococcus pyogenes (http://dna1.chem.uoknor.edu/strep.html); syn1, Synechocystis sp. PCC6803 (D90913). (B) gatA genes. afu1, Archaeoglobus fulgidus (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-tigrbl); bsu1, B. subtilis (AF008553); mca1, M. catarrhalis (U49269); mge1, Mycoplasma genitalium (U39689); mja1, Methanococcus jannaschii (U67558); mth1, Methanococcus thermoautotrophicum (22); mpn1, Mycoplasma pneumoniae (AE000058); ngo1, N. gonorrhoeae (http://dna1.chem.uoknor.edu/gono.html); rho1, Rhodococcus sp. N-774 (X54074); rho2, Rhodococcus sp. (M74531); sce1, Saccharomyces cerevisiae (L22072); spy1, S. pyogenes (http://dna1.chem.uo-knor.edu/strep.html); ssu1, Sulfolobus solfataricus (Y08256); syn1, Synechocystis sp. PCC6803 (D90913). (C) gatB genes. afu1, A. fulgidus (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-tigrbl); bsu1, B. subtilis 168 (AF008553); bsu2, B. subtilis 168M (U49790); eni1, Emericella nidulans (U62332). To condense the alignment figure, the N-terminal 57 amino acids (part of a mitochondrial targeting sequence) were omitted; mca1, M. catarrhalis (U49269), this sequence was edited in two places to generate a protein of 483 aa instead of the 419 aa protein reported; mge1, M. genitalium (U39689); mja1, M. jannaschii (U67473); mth1, M. thermoautotrophicum (22); mpn1, M. pneumoniae (AE000058); ngo1, N. gonorrhoeae (http://dna1.chem.uoknor.edu/gono.html); sce1, S. cerevisiae (D90913); spy1, S. pyogenes (http://dna1.chem.uoknor.edu/strep.html); syn1, Synechocystis sp. PCC6803 (D90913). The B. subtilis 168M clone shows only 75% amino acid identity with our B. subtilis 168 gatB gene. Because 168M is a cross of 168 with Bacillus W23, it may well be that the U49790 gatB gene originates from W23. This is likely, because the two gatB genes differ in their G+C content (bsu1, 46.5%; bsu2, 33.3%).

Figure 6

Alignment of gatA, gatB, and gatC genes with related sequences. Alignments were created with the Genetics Computer Group, clustalw, and boxshade programs. The database accession numbers are shown in parentheses. Red represents residues that are completely conserved in at least 85% of the sequences, blue those residues that are functionally conserved within the sequences, and black the other residues. (A) gatC genes. afu1, Archaeoglobus fulgidus (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-tigrbl); bsu1, B. subtilis (AF008553); mca1, M. catarrhalis (U49269); mja1, Methanococcus jannaschii (U67558) and also MJ0243; ngo1, Neisseria gonorrhoeae (http://dna1.chem.uoknor.edu/gono.html); sau1, Staphylococcus aureus (U06451); spy1, Streptococcus pyogenes (http://dna1.chem.uoknor.edu/strep.html); syn1, Synechocystis sp. PCC6803 (D90913). (B) gatA genes. afu1, Archaeoglobus fulgidus (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-tigrbl); bsu1, B. subtilis (AF008553); mca1, M. catarrhalis (U49269); mge1, Mycoplasma genitalium (U39689); mja1, Methanococcus jannaschii (U67558); mth1, Methanococcus thermoautotrophicum (22); mpn1, Mycoplasma pneumoniae (AE000058); ngo1, N. gonorrhoeae (http://dna1.chem.uoknor.edu/gono.html); rho1, Rhodococcus sp. N-774 (X54074); rho2, Rhodococcus sp. (M74531); sce1, Saccharomyces cerevisiae (L22072); spy1, S. pyogenes (http://dna1.chem.uo-knor.edu/strep.html); ssu1, Sulfolobus solfataricus (Y08256); syn1, Synechocystis sp. PCC6803 (D90913). (C) gatB genes. afu1, A. fulgidus (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-tigrbl); bsu1, B. subtilis 168 (AF008553); bsu2, B. subtilis 168M (U49790); eni1, Emericella nidulans (U62332). To condense the alignment figure, the N-terminal 57 amino acids (part of a mitochondrial targeting sequence) were omitted; mca1, M. catarrhalis (U49269), this sequence was edited in two places to generate a protein of 483 aa instead of the 419 aa protein reported; mge1, M. genitalium (U39689); mja1, M. jannaschii (U67473); mth1, M. thermoautotrophicum (22); mpn1, M. pneumoniae (AE000058); ngo1, N. gonorrhoeae (http://dna1.chem.uoknor.edu/gono.html); sce1, S. cerevisiae (D90913); spy1, S. pyogenes (http://dna1.chem.uoknor.edu/strep.html); syn1, Synechocystis sp. PCC6803 (D90913). The B. subtilis 168M clone shows only 75% amino acid identity with our B. subtilis 168 gatB gene. Because 168M is a cross of 168 with Bacillus W23, it may well be that the U49790 gatB gene originates from W23. This is likely, because the two gatB genes differ in their G+C content (bsu1, 46.5%; bsu2, 33.3%).