Methanogenic archaea possess unusual seryl-tRNA synthetase (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. The two types of SerRSs show only minimal sequence similarity, primarily within class II conserved motifs 1, 2 and 3. Here, we report a 2.5 Å resolution crystal structure of the atypical methanogenic Methanosarcina barkeri SerRS and its complexes with ATP, serine and the nonhydrolysable seryl-adenylate analogue 5 0 -O-(N-serylsulfamoyl)adenosine. The structures reveal two idiosyncratic features of methanogenic SerRSs: a novel N-terminal tRNA-binding domain and an active site zinc ion. The tetra-coordinated Zn 2 þ ion is bound to three conserved protein ligands (Cys306, Glu355 and Cys461) and binds the amino group of the serine substrate. The absolute requirement of the metal ion for enzymatic activity was confirmed by mutational analysis of the direct zinc ion ligands. This zinc-dependent serine recognition mechanism differs fundamentally from the one employed by the bacterial-type SerRSs. Consequently, SerRS represents the only known aminoacyl-tRNA synthetase system that evolved two distinct mechanisms for the recognition of the same amino-acid substrate.

Aminoacyl-tRNA synthetases (aaRSs) are ancient and evolutionary conserved enzymes catalyzing the formation of aminoacyl-tRNAs, that are used as substrates for ribosomal protein biosynthesis. In addition to full length aaRS genes, genomes of many organisms are sprinkled with truncated genes encoding single-domain aaRSlike proteins, which often have relinquished their canonical role in genetic code translation. We have identified the genes for putative seryl-tRNA synthetase homologs widespread in bacterial genomes and characterized three of them biochemically and structurally. The proteins encoded are homologous to the catalytic domain of highly diverged, atypical seryl-tRNA synthetases (aSerRSs) found only in methanogenic archaea and are deprived of the tRNA-binding domain. Remarkably, in comparison to SerRSs, aSerRS homologs display different and relaxed amino acid specificity. aSerRS homologs lack canonical tRNA aminoacylating activity and instead transfer activated amino acid to phosphopantetheine prosthetic group of putative carrier proteins, whose genes were identified in the genomic surroundings of aSerRS homologs. Detailed kinetic analysis confirmed that aSerRS homologs aminoacylate these carrier proteins efficiently and specifically. Accordingly, aSerRS homologs were renamed amino acid:[carrier protein] ligases (AMP forming). The enzymatic activity of aSerRS homologs is reminiscent of adenylation domains in nonribosomal peptide synthesis, and thus they represent an intriguing link between programmable ribosomal protein biosynthesis and template-independent nonribosomal peptide synthesis.

Aminoacyl-tRNA synthetases, a group of enzymes catalyzing aminoacyl-tRNA formation, may possess inherent editing activity to clear mistakes arising through the selection of non-cognate amino acid. It is generally assumed that both editing substrates, non-cognate aminoacyl-adenylate and misacylated tRNA, are hydrolyzed at the same editing domain, distant from the active site. Here, we present the first example of an aminoacyl-tRNA synthetase (seryl-tRNA synthetase) that naturally lacks an editing domain, but possesses a hydrolytic activity toward non-cognate aminoacyl-adenylates. Our data reveal that tRNA-independent pre-transfer editing may proceed within the enzyme active site without shuttling the non-cognate aminoacyl-adenylate intermediate to the remote editing site.

We have analyzed the evolution of recognition of tRNAsSerby seryl-tRNA synthetases, and compared it to other type 2 tRNAs, which contain a long extra arm. In Eubacteria and chloroplasts this type of tRNA is restricted to three families: tRNALeu, tRNASer and tRNATyr. tRNALeuand tRNASer also carry a long extra arm in Archaea, Eukarya and all organelles with the exception of animal mitochondria. In contrast, the long extra arm of tRNATyr is far less conserved: it was drastically shortened after the separation of Archaea and Eukarya from Eubacteria, and it is also truncated in animal mitochondria. The high degree of phylo-genetic divergence in the length of tRNA variable arms, which are recognized by both class I and class II aminoacyl-tRNA synthetases, makes type 2 tRNA recognition an ideal system with which to study how tRNA discrimination may have evolved in tandem with the evolution of other components of the translation machinery.

Two dissimilar seryl-transfer RNA (tRNA) synthetases (SerRSs) exist in Methanosarcina barkeri, one of bacterial type and the other resembling SerRSs present only in some methanogenic archaea. To investigate the requirements of these enzymes for tRNA Ser recognition, serylation of variant transcripts of M. barkeri tRNA Ser was kinetically analyzed in vitro with pure enzyme preparations. Characteristically for the serine system, the length of the variable arm was shown to be crucial for both enzymes, as was the identity of the discriminator base (G73). Moreover, a novel determinant for the specific tRNA Ser recognition was identified as the anticodon stem base pair G30:C40; its contribution to the efficiency of serylation was remarkable for both SerRSs. However, despite these similarities, the two SerRSs do not possess a uniform mode of tRNA Ser recognition, and additional determinants are necessary for serylation specificity by the methanogenic enzyme. In particular, the methanogenic SerRS relies on G1:C72 identity and on the number of unpaired nucleotides at the base of the variable stem for tRNA Ser recognition, unlike its bacterial type counterpart. We propose that such a distinction between the two enzymes in tRNA Ser identity determinants reflects their evolutionary pathways, hence attesting to their diversity.To maintain translational accuracy, aminoacyl-transfer RNA (tRNA) 1 synthetases are highly selective toward their amino acid and tRNA substrates. In the process of tRNA recognition, the cognate and non-cognate substrates are discriminated according to characteristic nucleotides in certain positions of the tRNA, specific for the tRNA/synthetase system.

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...

Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) contains a 20-amino acid C-terminal extension, which is not found in prokaryotic SerRS enzymes. A truncated yeast SES1 gene, lacking the 60 base pairs that encode this C-terminal domain, is able to complement a yeast SES1 null allele strain; thus, the C-terminal extension in SerRS is dispensable for the viability of the cell. However, the removal of the C-terminal peptide affects both stability of the enzyme and its affinity for the substrates. The truncation mutant binds tRNA with 3.6-fold higher affinity, while the K m for serine is 4-fold increased relative to the wild-type SerRS. This indicates the importance of the C-terminal extension in maintaining the overall structure of SerRS.Aminoacyl-tRNA synthetases are essential enzymes that catalyze the esterification of an amino acid to its cognate tRNA with exquisite specificity. The combination of biophysical, biochemical and genetic techniques have significantly deepened our understanding of the structure and function of these enzymes (1-3). The amino acid sequences of seryl-tRNA synthetases from Escherichia coli (4), Thermus thermophilus (5), Bacillus subtilis (6), Coxiella burnetii (7), Saccharomyces cerevisiae (8), Chinese hamster (partial) (9), and human 1 are known. According to common structural motifs they are typical representatives of class II synthetases (10). The crystal structures of two prokaryotic enzymes isolated from E. coli (11) and T. thermophilus (12) are quite similar, as is their mode of interaction with tRNA Ser . We have been working with yeast SerRS, 2 which shows only moderate similarity (about 30%) with prokaryotic seryl-tRNA synthetases on the level of the primary structure (5), but still recognizes bacterial tRNA Ser and can functionally substitute for the bacterial enzyme in vivo (13). The overexpression of the yeast SES1 gene in E. coli generated high amounts of functional enzyme (13), although with somewhat lower specific activity and slightly different electrophoretic mobility 3 compared to the enzyme isolated from yeast. This raises the possibility that the yeast enzyme may not be modified or folded correctly in the bacterial host. In this paper we describe the purification of yeast SerRS from an overproducing strain of S. cerevisiae. The alignment of the primary sequences of all SerRS proteins reveals that the enzymes from yeast (8), Chinese hamster (9), and human 1 contain C-terminal extension between 20 and 48 amino acids long not found in prokaryotic synthetases. We speculated whether this peptide was important for maintaining the structure of the eukaryotic enzymes or if it had another function. Thus, we deleted the part of the S. cerevisiae SES1 gene encoding the short C-terminal domain and analyzed the expressed truncated protein. (14). Selection for yeast auxotrophic markers was done in a medium of 0.67% nitrogen base and 2% glucose lacking amino acids, supplemented as needed with adenine (20 g/ml), uracil (20 g/ml), and amino acids (20 -30 g/ml). Sporulation mediu...

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.