Volume 28, Issue 2, Pages 228-239 (October 2007) The Transamidosome: A Dynamic Ribonucleoprotein Particle Dedicated to Prokaryotic tRNA-Dependent Asparagine Biosynthesis  Marc Bailly, Mickaël Blaise, Bernard Lorber, Hubert Dominique Becker, Daniel Kern  Molecular Cell  Volume 28, Issue 2, Pages 228-239 (October 2007) DOI: 10.1016/j.molcel.2007.08.017 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of the DRS2•tRNAAsn•AdT Complex by Gel Filtration (A) Gel filtration of free DRS2, AdT, and tRNAAsn. (B) Gel filtration of a DRS2 and tRNAAsn mix. (C) Gel filtration of an AdT and tRNAAsn mix. (D) Gel filtration of an AdT and DRS2 mix. (E) Gel filtration of a DRS2, tRNAAsn, and AdT mix. Gel filtrations were conducted as described in the Experimental Procedures with 10 μM of DRS2 subunits and/or 10 μM AdT and/or 10 μM tRNAAsn. The inserts show native PAGE of the mix. Molecular Cell 2007 28, 228-239DOI: (10.1016/j.molcel.2007.08.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Formation of a Ternary Complex with DRS2, AdT, and Free or Aspartylated or Asparaginylated tRNAAsn (A) Gel filtration of a mix of AdT, DRS2, tRNAAsn, and ATP. (B) Gel filtration of a mix of AdT, DRS2, and Asp-tRNAAsn. (C) Gel filtration of a mix of AdT, DRS2, and Asn-tRNAAsn. The gel filtrations were conducted as described in the Experimental Procedures with 10 μM of DRS2 subunits, 10 μM AdT, 20 μM free tRNAAsn, or [14C] Asp-tRNAAsn or [14C] Asn-tRNAAsn. The last pics from elution profiles represent part of ATP of the reaction mixture that coprecipitated by ethanol with the aa-tRNA. Molecular Cell 2007 28, 228-239DOI: (10.1016/j.molcel.2007.08.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Formation of the Transamidosome and Behavior during the Catalytic Process Monitored by Dynamic Light Scattering (A) Mean particle diameter of the free components. (B) Variation of the particle size of mixtures of DRS2 and increasing amounts of AdT; the apparent diameter increases by increasing the free AdT. (C)Variation of the particle size of mixtures of DRS2 and AdT with increasing amounts of tRNAAsn ; the apparent diameter decreases by increasing the free tRNAAsn. (D) Variation of the particle size during the catalytic steps; aspartylation of tRNAAsn by DRS leads to a reproducible decrease (shown by the arrow) of the particle size from 13.6 to 13.3 nm, whereas asparaginylation by amidation of tRNA-bound Asp into Asn increases it (shown by the arrow) from 13.3 to 13.5 nm. Molecular Cell 2007 28, 228-239DOI: (10.1016/j.molcel.2007.08.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Formation of the Transamidosome (A) Binding of tRNAAsn to DRS2. PAGE of mixtures containing 5000 cpm [32P]tRNAAsn and increasing amounts of DRS2. (B) Binding of tRNAAsn to AdT. PAGE of mixtures containing 10 μM of tRNAAsn and increasing amounts of AdT. (C) Binding of AdT to the DRS2•tRNAAsn complex. PAGE of mixtures containing 10 μM tRNAAsn, 10 μM DRS2 (4.5 μM of complex), and increasing amounts of AdT. (D) Saturation curves: AdT•tRNAAsn = f (AdT) obtained from (A) and DRS2•tRNAAsn•AdT = f(free AdT) obtained from (C). (E) The steps conducting to the ternary complex. Molecular Cell 2007 28, 228-239DOI: (10.1016/j.molcel.2007.08.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 The Two Pathways of Asn-tRNAAsn Formation (A) The indirect pathway: (1) Asp-tRNAAsn formation by free DRS2, (2) Asn-tRNAAsn formation from preformed Asp-tRNAAsn by free AdT. (B) The transamidosome-catalyzed pathway. The external arrows show the steps when the preformed transamidosome is incubated with the small ligands without free tRNAAsn: (1) formation of Asp-tRNAAsn, (2) formation of Asn-tRNAAsn from preformed Asp-tRNAAsn. The internal arrows represent the steps when the preformed transamidosome is incubated with all ligands in excess: (3) formation of the Asn-tRNAAsn at the presteady-state and (4) at the steady-state. The rate-limiting steps are shown by red arrows. (C) Kinetic of Asn-tRNAAsn formation when the preformed transamidosome is incubated with all ligands: (3) presteady state, (4) steady state. Molecular Cell 2007 28, 228-239DOI: (10.1016/j.molcel.2007.08.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Characterization of the Transamidosome by Gel Filtration of a Crude Protein Extract Isolated from an E. coli Strain Depending from tRNA-Dependent Transamidation Pathway to Form Asn-tRNAAsn (A) Gel filtration of the protein extract of the strain expressing D. radiodurans DRS2, N. meningitidis GatCAB, and T. thermophilus tRNAAsn. (B) Gel filtration of the protein extract of the strains expressing either DRS2 or GatCAB or tRNAAsn. Fifty microliters of the 105,000 × g supernatant was diluted in 100 μl of the gel filtration buffer and submitted to chromatography. The inserts show analysis by TLC of the tRNA-dependent conversion of Asp into Asn in presence of the fractions exhibiting the optimal aminoacylation and transamidation activities. The determinations of the catalytic activities of DRS2 and GatCAB, the measurements of the charging capacity of tRNAAsn and of the tRNA-dependent conversion of Asp into Asn and the TLC analysis were performed as described in the Experimental Procedures. Molecular Cell 2007 28, 228-239DOI: (10.1016/j.molcel.2007.08.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Docking Model of the Transamidosome (A) Side view of the model. The Thermus DRS2 (Charron et al., 2003; PDB: 1N9W) has been docked onto the tRNAAsp from yeast (PDB: 1ASY), and S. aureus GatCAB (Nakamura et al., 2006; PDB: 2G5H) onto the tRNAAsp acceptor arm. The DRS2 is in blue, the GatA, GatB, and GatC subunits in light orange, orange, and yellow, and the tRNA in gray. (B) Magnified view of the tRNA acceptor arm. The white arrow indicates the probable movement of the tRNA acceptor arm from DRS catalytic site toward the GatCAB active site. Molecular Cell 2007 28, 228-239DOI: (10.1016/j.molcel.2007.08.017) Copyright © 2007 Elsevier Inc. Terms and Conditions