SAXS analysis of the tRNA-modifying enzyme complex MnmE/MnmG reveals a novel interaction mode and GTP-induced oligomerization.

Marcus Fislage,Remy Loris,Egon Deyaert,Abel Garcia-Pino,Alessandro Spilotros,Elke Brosens,Els Pardon,Jan Steyaert,Wim Versées

doi:10.1093/nar/gku213

Abstract

Transfer ribonucleic acid (tRNA) modifications, especially at the wobble position, are crucial for proper and efficient protein translation. MnmE and MnmG form a protein complex that is implicated in the carboxymethylaminomethyl modification of wobble uridine (cmnm5U34) of certain tRNAs. MnmE is a G protein activated by dimerization (GAD), and active guanosine-5'-triphosphate (GTP) hydrolysis is required for the tRNA modification to occur. Although crystal structures of MnmE and MnmG are available, the structure of the MnmE/MnmG complex (MnmEG) and the nature of the nucleotide-induced conformational changes and their relevance for the tRNA modification reaction remain unknown. In this study, we mainly used small-angle X-ray scattering to characterize these conformational changes in solution and to unravel the mode of interaction between MnmE, MnmG and tRNA. In the nucleotide-free state MnmE and MnmG form an unanticipated asymmetric α2β2 complex. Unexpectedly, GTP binding promotes further oligomerization of the MnmEG complex leading to an α4β2 complex. The transition from the α2β2 to the α4β2 complex is fast, reversible and coupled to GTP binding and hydrolysis. We propose a model in which the nucleotide-induced changes in conformation and oligomerization of MnmEG form an integral part of the tRNA modification reaction cycle.

Highlights

Transfer ribonucleic acid molecules contain a vast number of modified nucleotides
We propose a model in which the nucleotide-induced changes in conformation and oligomerization of MnmE/MnmG complex (MnmEG) form an integral part of the Transfer ribonucleic acid (tRNA) modification reaction cycle
We have previously proposed that the large conformational changes of the G domains of MnmE upon GTP binding and hydrolysis are relayed throughout MnmE and MnmG and orchestrate the tRNA modification reaction [23,25]

Summary

Introduction

Transfer ribonucleic acid (tRNA) molecules contain a vast number of modified nucleotides. One of the main modification sites of tRNA is position 34, the so-called wobble position, that directly interacts with the third nucleotide of the messenger RNA (mRNA) codon. The proteins MnmE and MnmG form an enzyme complex (MnmEG) that is implicated in the modification of the wobble uridine in tRNALysmnm5s2UUU, tRNAGlumnm5s2UUC, tRNAGlncmnm5s2UUG, tRNALeucmnm5UmAA, tRNAArgmnm5UCU and tRNAGlymnm5UCC [5,6]. Except for the latter, all these tRNAs are reading A- and G-ending codons in split codon boxes [7,8]. Depending on the substrate that is being used, the MnmEG complex first forms either 5-carboxymethylaminomethyluridine

Methods

Results

Conclusion