Association between Archaeal Prolyl- and Leucyl-tRNA Synthetases Enhances tRNAPro Aminoacylation

Abstract

Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNA Pro charging showed that the catalytic efficiency (k cat /K m ) of ProRS increased 5-fold in the complex with LeuRS compared with the free enzyme, whereas the K m for proline was unchanged. No significant changes in the steady-state kinetics of LeuRS aminoacylation were observed upon the addition of ProRS. These findings indicate that ProRS and LeuRS associate in M. thermautotrophicus and suggest that this interaction contributes to translational fidelity by enhancing tRNA aminoacylation by ProRS.Aminoacyl-tRNA synthetases (aaRSs) 1 are essential components of the translation process. Their cellular role is to ensure that individual tRNAs are attached to their cognate amino acid. Each aaRS specifically binds to a defined set of tRNAs and catalyzes the attachment of the amino acids to the tRNAs. Once synthesized, the aminoacyl-tRNAs function as substrates for ribosomal protein synthesis, thereby ensuring correct translation of the genetic code (1). In bacteria, aaRSs typically perform their role as individual enzymes, found either as monomers, homodimers, or homo-or heterotetramers. However, in eukaryotes several aminoacyl-tRNA synthetases exist in multienzyme complexes (2-4), and two different types have so far been found in mammalian cells. One is composed of only one aminoacyl-tRNA synthetase, valyl-tRNA synthetase, and EF-1H, the heavy form of translation elongation factor 1 (5). The complex is believed to contain seven subunits, two monomeric subunits of valyl-tRNA synthetase and the EF-1H subunits EF1-␣, -␤, -␥, and -␦ in the molar ratio 2:1:1:1. The second complex described is considerably larger and includes nine aminoacyl-tRNA synthetase activities. The complex is composed of isoleucyl-, leucyl-(LeuRS), prolyl-(ProRS), methionyl-, glutaminyl-, glutamyl-, lysyl-, arginyl-, and aspartyl-tRNA synthetases. Whereas most of the aaRSs are present in the complex as monomers, data have indicated that lysyltRNA synthetase and aspartyl-tRNA synthetase exist as dimers (3,6). In addition, the polypeptide carrying the ProRS activity is multifunctional in that the protein also comprises the catalytic domain and activity of glutamyl-tRNA synthetase (7). Three auxiliary proteins, p18, p38, and p43, are also part of the multisynthetase complex. Although the structural and functional significance of the complex still remains to be elu...

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

“…Complex Formation between Archaeal MtSerRS and MtArgRS-AaRS aminoacylation efficiency can be increased through interactions with N-or C-terminal extensions in cis (49,50), and by the action of non-synthetase proteins and/or other synthetases acting in trans (21,27,32,40,41). Here we describe a new example of such an interaction, which appears to be particularly advantageous under adverse conditions; M. thermautotrophicus ArgRS, together with tRNA Arg , in a ternary complex with an atypical methanogenic-type SerRS (MtSerRS).…”

“…The cells were grown in medium without leucine (SDLeu) and were sequentially transformed with an M. thermautotrophicus cDNA library cloned into the vector pDEST22 (40), and all assays were performed according to the manufacturer's instructions (Invitrogen). Potential primary positives were selected by plating transformants on medium lacking leucine, tryptophan, and histidine (SD-Leu-Trp-His) supplemented with 25 mM 3-aminotriazole (3-AT) and the plates were incubated at 30°C for 3 days.…”

“…Yeast twohybrid screening identified 23 potentially positive interacting proteins with the MtSerRS bait, resulting in activation of reporter genes. The majority of interacting proteins identified from the screen were known false positives with broad interaction capability (MTH412, MTH674, MTH700, MTH913, MTH1168, MTH1878) (40,41) or other metabolic proteins (MTH846, MTH1107, MTH1134, MTH1738), based on previously published results for the same cDNA library (40,41).…”

“…Biophysical analyses confirmed the formation of a stable higher order archaeal MSC, highlighting the central role LeuRS plays as a core protein, mediating interactions within the complex. In this functional archaeal complex of four translational proteins, LeuRS was found to improve the catalytic efficiency of tRNA aminoacylation by both LysRS and ProRS (40). Furthermore, EF-1␣ increased k cat for Leu-tRNA Leu synthesis 8-fold, although complex formation had little effect on the activity of EF-1␣ itself (41).…”

Seryl-tRNA Synthetases in Translation and Beyond

“…[104] In Methanothermobacter thermautotrophicus, lysyl-tRNAsynthetase (LysRS) was found to be associated with leucyltRNA-synthetase (LeuRS) and prolyl-tRNA-synthetase (ProRS). [109][110][111] Besides, LeuRS forms a complex with elongation factor EF-1 alpha [100] adding this interaction to a relatively short list of associations found to be formed between EF-1 alpha and various aaRSs. [109,112] …”