The archaeal transamidosome for RNA-dependent glutamine biosynthesis

Abstract

Archaea make glutaminyl-tRNA (Gln-tRNA(Gln)) in a two-step process; a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) forms Glu-tRNA(Gln), while the heterodimeric amidotransferase GatDE converts this mischarged tRNA to Gln-tRNA(Gln). Many prokaryotes synthesize asparaginyl-tRNA (Asn-tRNA(Asn)) in a similar manner using a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) and the heterotrimeric amidotransferase GatCAB. The transamidosome, a complex of tRNA synthetase, amidotransferase and tRNA, was first described for the latter system in Thermus thermophilus [Bailly, M., Blaise, M., Lorber, B., Becker, H.D. and Kern, D. (2007) The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol. Cell, 28, 228-239.]. Here, we show a similar complex for Gln-tRNA(Gln) formation in Methanothermobacter thermautotrophicus that allows the mischarged Glu-tRNA(Gln) made by the tRNA synthetase to be channeled to the amidotransferase. The association of archaeal ND-GluRS with GatDE (K(D) = 100 ± 22 nM) sequesters the tRNA synthetase for Gln-tRNA(Gln) formation, with GatDE reducing the affinity of ND-GluRS for tRNA(Glu) by at least 13-fold. Unlike the T. thermophilus transamidosome, the archaeal complex does not require tRNA for its formation, is not stable through product (Gln-tRNA(Gln)) formation, and has no major effect on the kinetics of tRNA(Gln) glutamylation nor transamidation. The differences between the two transamidosomes may be a consequence of the fact that ND-GluRS is a class I aminoacyl-tRNA synthetase, while ND-AspRS belongs to the class II family.

Figure 1.

Formation of the M. thermautotrophicus GluRS:GatDE binary complex. Electrophoretic mobility shift assays were performed between purified GatDE and ND-aaRS indicated as described in the ‘Materials and Methods’ section. Native PAGE (10%) separation of samples containing: (A) a stable concentration of GatDE (1 µM) and increasing concentrations of ND-GluRS (0–10 µM), (B) GatDE (3 µM) alone (lanes 1, 3 and 5) or both GatDE and GluRS (3 µM each) (lanes 2, 4 and 6) incubated at the temperatures indicated, and (C) a stable concentration of GatDE (1 µM) and increasing concentrations of ND-AspRS (0–10 µM), stained with Coomassie blue. (D) Ni-NTA pull-down with His₆-tagged GatDE. Proteins were separated by SDS–PAGE and AF-GluRS was visualized by UV illumination. Escherichia coli extracts from cells either over-expressing His₆-tagged GatDE (OE; lanes 3 and 4) or not (No; lane 2), were incubated with (+) or without (−) AF-GluRS (0.8 mg), and Ni-NTA agarose beads followed by three washes as described in the ‘Materials and Methods’ section. Purified AF-GluRS prior to Ni-NTA pull-down run in lane 1.

Figure 2.

Gel filtration analysis of the archaeal GatDE:GluRS binary complex. Gel filtrations over a sephacryl S300 16/60 column were carried out as described in the ‘Materials and Methods’ section with GatDE (10 µM) and/or ND-GluRS (20 µM) added to the mix. Gel filtration analysis of (A) GatDE alone, (B) ND-GluRS alone and (C) GatDE and ND-GluRS pre-incubated together. (D) Combined aminoacylation/amidotransferase assay with ³²P-labeled tRNA^Gln (100 nM), ATP (4 mM), l-Glu (2 mM) and Asn (2 mM) incubated with (lane 1) no enzyme, (lane 2) purified GatDE (250 nM), (Lane 3) purified ND-GluRS (250 nM), (Lane 4) purified ND-GluRS and GatDE (250 nM each) or (lane 5) the ND-GluRS:GatDE complex (400 nM) eluted from the gel filtration column (Figure 2C). (E) Calibration curve with known molecular weight standards (filled circle) and the ND-GluRS:GatDE complex (open circle).

Figure 3.

Formation of the ND-GluRS:GatDE:tRNA^Gln complex. Electrophoretic mobility shift assays were performed between purified ND-GluRS and GatDE with ³²P-labeled tRNA^Gln or tRNA^Glu as described in the ‘Materials and Methods’ section. (A) ³²P-labeled tRNA^Gln (8 µM) incubated with no enzyme (lane 1) or increasing concentrations of ³²P-labeled tRNA^Gln incubated with GatDE (1 µM; lanes 2–8). (B) ³²P-labeled tRNA^Gln (100 nM) incubated with (lane 1) no enzyme, (lane 2) GatDE (6.0 µM), (lane 3) ND-GluRS (1 µM) or (lanes 4–9) ND-GluRS (1 µM) and GatDE (1.0–6.0 µM). (C) ³²P-labeled tRNA^Glu (100 nM) incubated with (lane 1) no enzyme, (lane 2) ND-GluRS (1 µM), (lane 3) GatDE (6 µM) or (lanes 4–7) ND-GluRS (1 µM) and GatDE (1.0–6.0 µM).

Figure 4.

Gel filtration analysis of the archaeal GatDE:GluRS:tRNA^Gln complex. Gel filtrations over a sephacryl S300 16/60 column were carried out as described in the ‘Materials and Methods’ section with tRNA^Gln (20 µM) and protein(s) indicated (GatDE and/or ND-GluRS) (20 µM). (A) Gel filtration and native PAGE analysis of free tRNA^Gln. (B) Gel filtration and native PAGE analysis of a sample containing GatDE with tRNA^Gln. The arrow indicates how long approximately GatDE is retained on the column (Figure 2A). (C) Gel filtration and native PAGE analysis of a sample containing GluRS and tRNA^Gln. (D) Gel filtration and native PAGE analysis of a sample containing GluRS, GatDE and tRNA^Gln.

Figure 5.

Gel filtration analysis after aminoacylation and transamidation. Gel filtrations over a sephacryl S300 16/60 column were carried out as described in the ‘Materials and Methods’ section with tRNA^Gln, GatDE and ND-GluRS, (20 µM each) and either (A) ATP (4 mM) and l-Glu (3.5 mM), or (B) ATP (4 mM), l-Glu (3.5 mM) and Asn (4 mM) added to the incubation mixture.

Figure 6.

Cycle of RNA-dependent biosynthesis of Gln in Archaea. (1) GatDE (orange) associates with ND-GluRS (lime). (2) ND-GluRS:GatDE binary complex binds tRNA^Gln (silver-blue) to form the ternary complex. (3) In the transamidosome (ND-GluRS:GatDE:tRNA^Gln), ND-GluRS glutamylates tRNA^Gln. (4) ND-GluRS dissociates from the complex allowing the 3′ CCA-end of the tRNA to flip into the GatDE kinase active site. (5) GatDE transamidates the tRNA-bound Glu to Gln. (6) Gln-tRNA^Gln is released from GatDE. For clarity only one GatDE monomer is shown.