A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

Cindy L Wolfe,J Anthony Warrington,Lauren Treadwell,Mona Trempe Norcum

doi:10.1074/jbc.m502759200

Cindy L Wolfe, J Anthony Warrington + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m502759200

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Abstract

It has become evident that the process of protein synthesis is performed by many cellular polypeptides acting in concert within the structural confines of protein complexes. In multicellular eukaryotes, one of these assemblies is a multienzyme complex composed of eight proteins that have aminoacyl-tRNA synthetase activities as well as three non-synthetase proteins (p43, p38, and p18) with diverse functions. This study uses electron microscopy and three-dimensional reconstruction to explore the arrangement of proteins and tRNA substrates within this "core" multisynthetase complex. Binding of unfractionated tRNA establishes that these molecules are widely distributed on the exterior of the structure. Binding of gold-labeled tRNA(Leu) places leucyl-tRNA synthetase and the bifunctional glutamyl-/prolyl-tRNA synthetase at the base of this asymmetric "V"-shaped particle. A stable cell line has been produced that incorporates hexahistidine-labeled p43 into the multisynthetase complex. Using a gold-labeled nickel-nitrilotriacetic acid probe, the polypeptides of the p43 dimer have been located along one face of the particle. The results of this and previous studies are combined into an initial three-dimensional working model of the multisynthetase complex. This is the first conceptualization of how the protein constituents and tRNA substrates are arrayed within the structural confines of this multiprotein assembly.

Highlights

This is a intriguing protein assembly as all of the enzymes catalyze the same reaction, albeit with individual and highly specific substrates
By use of its cognate tRNA labeled with a gold reporter molecule, the position of the LeuRS active site is placed at a specific location within the three-dimensional structure of the human multisynthetase complex as calculated from electron microscopic images
Positioning of Multiple Bound tRNAs—As a way to obtain a general idea of how the multisynthetase complex accommodates the simultaneous binding of multiple tRNAs, a reconstruction was calculated after reaction with a mixture of tRNAs

Summary

Introduction

This is a intriguing protein assembly as all of the enzymes catalyze the same reaction, albeit with individual and highly specific substrates. The particle is shaped as an asymmetric “V.” Its most striking features are a deep central cleft and multiple “windows” into the interior These reconstructions are of sufficient resolution to show distinct protrusions and sub-domains of the particle, which are likely to correspond to individual proteins within the structure. Such information in turn is fundamental in providing answers to the questions posed above regarding how the multisynthetase complex works This initial approach examines the distribution of tRNA binding sites on the particle and uses this information to position the corresponding aminoacyl-tRNA synthetases. By use of its cognate tRNA labeled with a gold reporter molecule, the position of the LeuRS active site is placed at a specific location within the three-dimensional structure of the human multisynthetase complex as calculated from electron microscopic images. This study describes the first incorporation of a tagged protein component into the particle

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