title: Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where GluRS2 hydrolyzes excess Glu-tRNAGln.
abstract: In many bacteria and archaea, an ancestral pathway is used where asparagine and glutamine are formed from their acidic precursors while covalently linked to tRNA(Asn) and tRNA(Gln), respectively. Stable complexes formed by the enzymes of these indirect tRNA aminoacylation pathways are found in several thermophilic organisms, and are called transamidosomes. We describe here a transamidosome forming Gln-tRNA(Gln) in Helicobacter pylori, an ε-proteobacterium pathogenic for humans; this transamidosome displays novel properties that may be characteristic of mesophilic organisms. This ternary complex containing the non-canonical GluRS2 specific for Glu-tRNA(Gln) formation, the tRNA-dependent amidotransferase GatCAB and tRNA(Gln) was characterized by dynamic light scattering. Moreover, we observed by interferometry a weak interaction between GluRS2 and GatCAB (K(D) = 40 ± 5 µM). The kinetics of Glu-tRNA(Gln) and Gln-tRNA(Gln) formation indicate that conformational shifts inside the transamidosome allow the tRNA(Gln) acceptor stem to interact alternately with GluRS2 and GatCAB despite their common identity elements. The integrity of this dynamic transamidosome depends on a critical concentration of tRNA(Gln), above which it dissociates into separate GatCAB/tRNA(Gln) and GluRS2/tRNA(Gln) complexes. Ester bond protection assays show that both enzymes display a good affinity for tRNA(Gln) regardless of its aminoacylation state, and support a mechanism where GluRS2 can hydrolyze excess Glu-tRNA(Gln), ensuring faithful decoding of Gln codons.
authors.affiliation: Département de Biochimie, de Microbiologie et de Bio-informatique, PROTEO et IBIS, Université Laval, 1045 av de la Médecine, Québec, Québec, Canada.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Huot
authors.forename: Frédéric
authors.initials: F
authors.lastname: Fischer
authors.forename: Jacques
authors.initials: J
authors.lastname: Corbeil
authors.forename: Eric
authors.initials: E
authors.lastname: Madore
authors.forename: Bernard
authors.initials: B
authors.lastname: Lorber
authors.forename: Guillaume
authors.initials: G
authors.lastname: Diss
authors.forename: Tamara L
authors.initials: TL
authors.lastname: Hendrickson
authors.forename: Daniel
authors.initials: D
authors.lastname: Kern
authors.forename: Jacques
authors.initials: J
authors.lastname: Lapointe
mesh_terms: D005975:Glutamate-tRNA Ligase / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007368:Interferometry; D007700:Kinetics; D008954:Models, Biological; D019884:Nitrogenous Group Transferases / Q000378:metabolism*; D020871:RNA Stability; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012351:RNA, Transfer, Gln / Q000378:metabolism*
chemical_list: D012346:RNA, Transfer, Amino Acyl; D012351:RNA, Transfer, Gln; C525150:GatCAB amidotransferase, Helicobacter pylori; D019884:Nitrogenous Group Transferases; D005975:Glutamate-tRNA Ligase