A tRNA-independent Mechanism for Transamidosome Assembly Promotes Aminoacyl-tRNA Transamidation

Gayathri N Silva,Shirin Fatma,Ashley M Floyd,Frederic Fischer,Pitak Chuawong,Amanda N Cruz,Rachel M Simari,Nilesh Joshi,Daniel Kern,Tamara L Hendrickson

doi:10.1074/jbc.m112.441394

Abstract

Many bacteria lack genes encoding asparaginyl- and/or glutaminyl-tRNA synthetase and consequently rely on an indirect path for the synthesis of both Asn-tRNA(Asn) and Gln-tRNA(Gln). In some bacteria such as Thermus thermophilus, efficient delivery of misacylated tRNA to the downstream amidotransferase (AdT) is ensured by formation of a stable, tRNA-dependent macromolecular complex called the Asn-transamidosome. This complex enables direct delivery of Asp-tRNA(Asn) from the non-discriminating aspartyl-tRNA synthetase to AdT, where it is converted into Asn-tRNA(Asn). Previous characterization of the analogous Helicobacter pylori Asn-transamidosome revealed that it is dynamic and cannot be stably isolated, suggesting the possibility of an alternative mechanism to facilitate assembly of a stable complex. We have identified a novel protein partner called Hp0100 as a component of a stable, tRNA-independent H. pylori Asn-transamidosome; this complex contains a non-discriminating aspartyl-tRNA synthetase, AdT, and Hp0100 but does not require tRNA(Asn) for assembly. Hp0100 also enhances the capacity of AdT to convert Asp-tRNA(Asn) into Asn-tRNA(Asn) by ∼35-fold. Our results demonstrate that bacteria have adopted multiple divergent methods for transamidosome assembly and function.

Highlights

Some microorganisms use indirect tRNA aminoacylation to produce Asn-tRNAAsn; the necessary components are assembled into a tRNAAsn-dependent transamidosome complex
This transamidosome assembles from an archaeal-type ND-AspRS, tRNAAsn, and AdT; two of the four tRNAs bound to this complex are structural, making this complex a tRNA-containing ribonucleoprotein complex [12]
We have demonstrated that the H. pylori Asn-transamidosome is not a tRNA-containing ribonucleoprotein complex because the catalytically competent complex assembles in the absence of tRNAAsn, relegating this tRNA to the role of substrate only

Summary

Background

Some microorganisms use indirect tRNA aminoacylation to produce Asn-tRNAAsn; the necessary components are assembled into a tRNAAsn-dependent transamidosome complex. In some bacteria such as Thermus thermophilus, efficient delivery of misacylated tRNA to the downstream amidotransferase (AdT) is ensured by formation of a stable, tRNA-dependent macromolecular complex called the Asn-transamidosome This complex enables direct delivery of Asp-tRNAAsn from the nondiscriminating aspartyl-tRNA synthetase to AdT, where it is converted into Asn-tRNAAsn. Previous characterization of the analogous Helicobacter pylori Asn-transamidosome revealed that it is dynamic and cannot be stably isolated, suggesting the possibility of an alternative mechanism to facilitate assembly of a stable complex. We have identified a novel protein partner called Hp0100 as a component of a stable, tRNA-independent H. pylori Asn-transamidosome; this complex contains a nondiscriminating aspartyl-tRNA synthetase, AdT, and Hp0100 but does not require tRNAAsn for assembly. Hp0100 simplifies the process of indirect tRNA aminoacylation by bringing all enzymatic players into proximity in a distinct Asn-transamidosome

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION