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

Abstract

Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation, although the precise biological role remains largely unknown. To gain further insights into archaeal MSCs, possible proteinprotein interactions with the atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS) were investigated. Yeast two-hybrid analysis revealed arginyl-tRNA synthetase (MtArgRS) as an interacting partner of MtSerRS. Surface plasmon resonance confirmed stable complex formation, with a dissociation constant (K D ) of 250 nM. Formation of the MtSerRS⅐MtArgRS complex was further supported by the ability of GST-MtArgRS to co-purify MtSerRS and by coelution of the two enzymes during gel filtration chromatography. The MtSerRS⅐MtArgRS complex also contained tRNA Arg , consistent with the existence of a stable ribonucleoprotein complex active in aminoacylation. Steady-state kinetic analyses revealed that addition of MtArgRS to MtSerRS led to an almost 4-fold increase in the catalytic efficiency of serine attachment to tRNA, but had no effect on the activity of MtArgRS. Further, the most pronounced improvements in the aminoacylation activity of MtSerRS induced by MtArgRS were observed under conditions of elevated temperature and osmolarity. These data indicate that formation of a complex between MtSerRS and MtArgRS provides a means by which methanogenic archaea can optimize an early step in translation under a wide range of extreme environmental conditions. Aminoacyl-tRNA synthetases (aaRSs)2 catalyze the specific coupling of amino acids with their cognate tRNAs to produce aminoacyl-tRNAs (aa-tRNAs), which serve as starting materials for the biosynthesis of proteins. Aa-tRNA synthesis occurs in two steps: amino acid activation at the expense of ATP followed by the aminoacylation of tRNA (1). Although for most aaRSs the formation of aminoacyl-AMP does not require tRNA, cognate tRNA is necessary for amino acid activation by ArgRS, GlnRS, GluRS, and LysRS1 enzymes from many organisms (2). Based on structural features of their active sites, aaRSs can be divided into two classes, which comprise 10 members each (3). In addition, an unusual form of LysRS is found in class I (2), while class II also includes the noncanonical synthetases PylRS and SepRS (4).In all three domains of life, subsets of aaRSs have been shown to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSCs). These complexes are distinctive compared with other macromolecular protein complexes, because their components are enzymes that carry out similar catalytic reactions simultaneously, and only some aaRSs are involved (5). In eukaryotes, MSCs tend to be larger than those discovered in bacteria and archaea and also perform a wider range of functions that include both aminoacylation and noncanonical roles b...

Seryl-tRNA Synthetases in Translation and Beyond

“…Using the Y2H methodology we found that the archaeal SerRS can interact with ribosomal proteins and ArgRS. [113] We found that the key functional outcome of the complex formation between ArgRS and SerRS in archaea is its stimulating effect on the rate of serylation. The optimal activity of SerRS is achieved in a complex with ArgRS at 200-300 mM NaCl and at the temperature of 65 °C which is in accordance with the growth of M. thermautotrophicus under elevated osmolarity and temperature.…”

“…Our data support the notion that SerRS:ArgRS complex may constitute a part of the thermoand osmoadaptation mechanisms of thermophilic methanogenic archaea, by providing an optimal microenvironment for the tRNA aminoacylation under a wide range of conditions. [113] Beside ArgRS, the Y2H screen identified the ribosomal protein L3 as a SerRS interactor, indicating a possible interaction of the archaeal aaRSs with the ribosome. In further characterization of interactions of archaeal aaRSs with the ribosome we showed by microscale thermophoresis (MST) that atypical SerRS and an archaeal ArgRS bind to the large ribosomal subunit with micromolar affinities.…”

How to Study Protein-protein Interactions

“…This step largely depends on dissociation rates. 39 Besides proteins it is possible to immobilize various lipid membrane systems and in this way study protein-membrane interactions and even elucidate mechanisms of pore formation for many important molecules. [40][41][42] The method is often applied in drug discovery, since the technology has evolved enormously towards high-throughput instrumentation.…”

Arginyl-tRNA Synthetase Facilitates Complex Formation Between Seryl-tRNA Synthetase and its Cognate Transfer RNA

“…Both types of enzymes bind one or two cognate tRNAs across two protein subunits, 12,15 and it seems that in both systems tRNA binding is enhanced by the interaction of SerRSs with other proteins. 6,16 We have previously shown that yeast SerRS (a bacterial-type enzyme) associates with peroxin Pex21p, 16 while SerRSs from methanogens form assemblies with arginyl-tRNA synthetase (ArgRS). 6 In this paper we present further evidence that interaction of MtSerRS and tRNA…”

“…In this context, we previously explored the ability of seryl-tRNA synthetases (SerRSs), which catalyze esterification of cognate tRNA Ser isoacceptors with serine, to interact with other aaRSs and with nonsynthetase proteins. 6 Although, generally, the aaRSs that recognize the same amino acid are rather conserved in terms of sequences and 3D-structures in all domains of life, the two diverged types of SerRSs exist. [7][8][9][10] Bacterial-type SerRSs function in a variety of archaeal, bacterial and eukaryotic organisms, while somewhat atypical, methanogenic-type SerRS was found only in methanogenic archaea, 10,11 the organisms which often inhabit the environments characterized by extreme living conditions (anaerobic, thermofilic, psychrophilic, halophilic).…”