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 tRNAAsn and tRNAGln, 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-tRNAGln 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-tRNAGln formation, the tRNA-dependent amidotransferase GatCAB and tRNAGln was characterized by dynamic light scattering. Moreover, we observed by interferometry a weak interaction between GluRS2 and GatCAB (KD = 40 ± 5 µM). The kinetics of Glu-tRNAGln and Gln-tRNAGln formation indicate that conformational shifts inside the transamidosome allow the tRNAGln 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 tRNAGln, above which it dissociates into separate GatCAB/tRNAGln and GluRS2/tRNAGln complexes. Ester bond protection assays show that both enzymes display a good affinity for tRNAGln regardless of its aminoacylation state, and support a mechanism where GluRS2 can hydrolyze excess Glu-tRNAGln, ensuring faithful decoding of Gln codons.

Ancient translation factor is essential for tRNA-dependent cysteine biosynthesis in methanogenic archaea

“…(ii) The Gln and Asn transamidosomes in Helicobacter pylori are transiently formed in the presence of tRNA Gln and tRNA Asn , respectively (32,33). A protein component Hp0100 can facilitate the formation of a stable Asn transamidosome in H. pylori (34).…”

Structure of the Pseudomonas aeruginosa transamidosome reveals unique aspects of bacterial tRNA-dependent asparagine biosynthesis

“…With the tRNA Asn 3′ end bound in the GatCAB active site, the PaAsn-transamidosome likely represents the transamidation state of the complex. Apart from association with ND-AspRS, bacterial GatCAB can form a ternary complex with ND-GluRS and tRNA Gln , known as a Gln-transamidosome and typical of organisms lacking a GlnRS (30,31). In the Thermotoga maritima (Tm) Gln-transamidosome structure (30), the tRNA Gln 3′ end is bound in the GluRS active site, and the GatCAB tail domain is associated with the D-loop of the tRNA.…”

Glutamyl-tRNA in Bacteria. Multiple Identities for Multiple Functions

“…35,48,50,56 In these complexes, the AdT must unambiguously recognize GlutRNA Gln and Asp-tRNA Asn instead of the correctly charged Glu-tRNA Glu and Asp-tRNA Asp . In the complex the two enzymes cannot use the same identity elements simultaneously.…”

“…Also it accounts for the fact that the tRNA has to easily leave the complex to enter protein translation. 50 Crystal structures of transamidosomes from thermophiles have been obtained, 35,56 although in the case of the complex from T. maritima it was necessary to covalently link NDGluRS to GatC in order to obtain structural data. 35 Interestingly, the general structure of transdamidosomes GluRS-GatCAB from T. maritima and AspRS-GatCAB from T. thermophilus differ considerably.…”