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...

Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6 Å resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea.

Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.