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

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 tRNALeu 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.It has been known for many years that a "core" complex of aminoacyltRNA synthetases can be isolated from all multicellular eukaryotes (1). Its characteristic composition is three non-synthetase proteins and eight polypeptides having nine aminoacyl-tRNA synthetase activities. With the enzymes abbreviated as the three-letter amino acid name plus RS for the enzyme, the components are ArgRS, AspRS, GluProRS, GlnRS, IleRS, LeuRS, LysRS, MetRS, p43, p38, and p18. Several of these are known dimers, so the total polypeptide count in the multisynthetase complex is at least fifteen. This is a particularly intriguing protein assembly as all of the enzymes catalyze the same reaction, albeit with individual and highly specific substrates. The reaction catalyzed is the covalent attachment of an amino acid to either the 2Ј-or 3Ј-hydroxyl of the 3Ј-terminal adenosine of tRNA. Recent increased interest in these enzymes has been generated by studies indicating that they have roles in a variety of other cellular processes (2-4). Such activities range from cytokine-mediated chemoattraction to priming of reverse transcription of the human immunodeficiency virus, type 1 genome. These enzymes are also viewed as targets in the development of new antimicrobial agents (5). The multisynthetase complex also contains three non-synthetase proteins. The first of these, p18, has been shown to interact with EF-1␥, which may facilitate transfer of aminoacyl-tRNAs to the ribosome (6). Data from yeast two-hybrid screens (7), in vitro binding assays (8), and deletion analysis (9) indicate that p38 functions as a scaffolding protein and appears to be...

Structural context for mobilization of a human tRNA synthetase from its cytoplasmic complex

“…6) (24,25). These findings further define the composition of MSC, in which most components were proposed to have a stoichiometry of one per MSC (31,(43)(44)(45). Close inspection of the published work on previously purified MSCs, from various species, further indicates that, in many preparations, LysRS has twice the density expected for two LysRS molecules per MSC (24,31,46,47).…”

“…The critical role of LysRS for the assembly and stability of the MSC underscores the importance of maintaining a basal level of LysRS within the MSC, even when it is specifically released in response to external or internal stimulations. Indeed, some variance in the stoichiometry of binding of LysRS to the MSC has been reported (43,45,49).…”

DARS -associated leukoencephalopathy can mimic a steroid-responsive neuroinflammatory disorder

“…Intriguingly, recent structural data have demonstrated a similar 3D structure of both cytosolic and mitochondrial AspRS despite poor sequence homology of their respective genes, raising the possibility of functional overlap in situ, which might provide clues to the striking parallels between LBSL and HBSL. 9 Cytosolic AspRS is part of the multisynthetase complex, 10 which appears to play an important role not only in tRNA synthesis, but also in multiple cellular processes, including cytokine and immune responses. 11 The apparent positive response of several patients with HBSL to anti-inflammatory treatment might be related to these alternative functions.…”

An Archaeal tRNA-Synthetase Complex that Enhances Aminoacylation under Extreme Conditions

“…This complex is composed of eleven polypeptides including the bi-functional glutamyl-prolyl-tRNA synthetase (GluProRS), seven monospecific aspartyl-, arginyl-, glutaminyl-, lysyl-, methionyl-, leucyl-, and isoleucyl-tRNA synthetases (AspRS, ArgRS, GlnRS, LysRS, MetRS, LeuRS, and IleRS, respectively); and three nonsynthetase protein factors, p18, p38, and p43 (20,21,22). The structural organization of this complex has not yet been completely deciphered (23,24), but the stability of some components has been shown to depend on their proximity to neighboring proteins (25). The other known multiprotein mammalian aaRS complex comprises just two monomeric subunits of valyl-tRNA synthetase and several subunits of translation elongation factor EF-1H (26).…”