Sequence Divergence of Seryl-tRNA Synthetases in Archaea

The genomic sequences of Methanococcus jannaschii and Methanobacterium thermoautotrophicum contain a structurally uncommon seryl-tRNA synthetase (SerRS) sequence and lack an open reading frame (ORF) for the canonical cysteinyl-tRNA synthetase (CysRS). Therefore, it is not clear if Cys-tRNA^Cys is formed by direct aminoacylation or by a transformation of serine misacylated to tRNA^Cys. To address this question, we prepared SerRS from two methanogenic archaea and measured the enzymatic properties of these proteins. SerRS was purified from M. thermoautotrophicum; its N-terminal peptide sequence matched the sequence deduced from the relevant ORF in the genomic data of M. thermoautotrophicum and M. jannaschii. In addition, SerRS was expressed from a cloned Methanococcus maripaludis serS gene. The two enzymes charged serine to their homologous tRNAs and also accepted Escherichia coli tRNA as substrate for aminoacylation. Gel shift experiments showed that M. thermoautotrophicum SerRS did not mischarge tRNA^Cys with serine. This indicates that Cys-tRNA^Cys is formed by direct acylation in these organisms.     

Recognition between tRNASer and archaeal seryl-tRNA synthetases monitored by suppression of bacterial amber mutations

Two dissimilar seryl-tRNA synthetases (SerRSs) exist in Methanosarcina barkeri : one of bacterial type (bMbSerRS) and the other resembling SerRSs present only in methanogenic archaea (mMbSerRS). While the expression of the archaeal bMbSerRS gene in Escherichia coli complements the function of thermolabile SerRS at a nonpermissive temperature, mMbSerRS does not. Our recent X-ray structural analysis of mMbSerRS revealed an idiosyncratic N-terminal domain and a catalytic zinc ion in the active site, identifying methanogenic-type SerRSs as atypical members of the SerRS family. To shed further light on substrate discrimination by methanogenic-type SerRS, we developed an in vivo system in E. coli to study tRNA serylation by mMbSerRS variants. We show that coexpression of the M. barkeri SerRS gene, encoding either bacterial- or methanogenic-type SerRS, with the gene for cognate archaeal suppressor tRNA leads to suppression of bacterial amber mutations, implying that the E. coli translation machinery can use serylated tRNA from methanogenic archaea as a substrate in protein synthesis. Furthermore, because serylation of M. barkeri serine-specific tRNA by endogenous E. coli SerRS is negligible, suppression is entirely dependent on recognition between archaeal partners (mMbSerRS/suppressor tRNA^Ser). Thus, the efficiency of suppression by mMbSerRS variants quantified in the described β-galactosidase-based reporter system, accurately reflects enzymes' serylation propensity obtained by in vitro kinetic measurements.     

tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase.

The ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code. Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) employs tRNA-dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., S?ll, D. & Weygand-Durasevic, I. (1997) J. Biol. Chem.272, 1136-1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl-adenylate formation than the apoenzyme alone. Sequence-specific tRNA-protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA-induced conformational change in the enzyme's active site. The 3'-terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA-assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.     

Biosynthesis of phosphoserine in the Methanococcales

Methanococcus maripaludis and Methanocaldococcus jannaschii produce cysteine for protein synthesis using a tRNA-dependent pathway. These methanogens charge tRNA(Cys) with l-phosphoserine, which is also an intermediate in the predicted pathways for serine and cystathionine biosynthesis. To establish the mode of phosphoserine production in Methanococcales, cell extracts of M. maripaludis were shown to have phosphoglycerate dehydrogenase and phosphoserine aminotransferase activities. The heterologously expressed and purified phosphoglycerate dehydrogenase from M. maripaludis had enzymological properties similar to those of its bacterial homologs but was poorly inhibited by serine. While bacterial enzymes are inhibited by micromolar concentrations of serine bound to an allosteric site, the low sensitivity of the archaeal protein to serine is consistent with phosphoserine's position as a branch point in several pathways. A broad-specificity class V aspartate aminotransferase from M. jannaschii converted the phosphohydroxypyruvate product to phosphoserine. This enzyme catalyzed the transamination of aspartate, glutamate, phosphoserine, alanine, and cysteate. The M. maripaludis homolog complemented a serC mutation in the Escherichia coli phosphoserine aminotransferase. All methanogenic archaea apparently share this pathway, providing sufficient phosphoserine for the tRNA-dependent cysteine biosynthetic pathway.     

Mytilus mitochondrial DNA contains a functional gene for a tRNASer(UCN) with a dihydrouridine arm-replacement loop and a pseudo-tRNASer(UCN) gene.

A 2500-nucleotide pair (ntp) sequence of F-type mitochondrial (mt) DNA of the Pacific Rim mussel Mytilus californianus (class Bivalvia, phylum Mollusca) that contains two complete (ND2 and ND3) and two partial (COI and COIII) protein genes and nine tRNA genes is presented. Seven of the encoded tRNAs (Ala, Arg, His, Met(AUA), Pro, Ser(UCN), and Trp) have the potential to fold into the orthodox four-armed tRNA secondary structure, while two [tRNASer(AGN) and a second tRNASer(UCN)] will fold only into tRNAs with a dihydrouridine (DHU) arm-replacement loop. Comparison of these mt-tRNA gene sequences with previously published, corresponding M. edulis F-type mtDNA indicates that similarity between the four-armed tRNASer(UCN) genes is only 63.8% compared with an average of 92.1% (range 86.2-98. 5%) for the remaining eight tRNA genes. Northern blot analysis indicated that mature tRNAs encoded by the DHU arm-replacement loop-containing tRNASer(UCN), tRNASer(AGN), tRNAMet(AUA), tRNATrp, and tRNAPro genes occur in M. californianus mitochondria, strengthening the view that all of these genes are functional. However, Northern blot and 5' RACE (rapid amplification of cDNA ends) analyses indicated that the four-armed tRNASer(UCN) gene is transcribed into a stable RNA that includes the downstream COI sequence and is not processed into a mature tRNA. On the basis of these observations the M. californianus and M. edulis four-armed tRNASer(UCN) sequences are interpreted as pseudo-tRNASer(UCN) genes.     

Selective inhibition of divergent seryl-tRNA synthetases by serine analogues

Seryl-tRNA synthetases (SerRSs) fall into two distinct evolutionary groups of enzymes, bacterial and methanogenic. These two types of SerRSs display only minimal sequence similarity, primarily within the class II conserved motifs, and possess distinct modes of tRNA(Ser) recognition. In order to determine whether the two types of SerRSs also differ in their recognition of the serine substrate, we compared the sensitivity of the representative methanogenic and bacterial-type SerRSs to serine hydroxamate and two previously unidentified inhibitors, serinamide and serine methyl ester. Our kinetic data showed selective inhibition of the methanogenic SerRS by serinamide, suggesting a lack of mechanistic uniformity in serine recognition between the evolutionarily distinct SerRSs.     

An idiosyncratic serine ordering loop in methanogen seryl-tRNA synthetases guides substrates through seryl-tRNASer formation

Seryl-tRNA synthetases (SerRS) covalently attach serine to cognate tRNA^Ser. Atypical SerRSs, considerably different from canonical enzymes, have been found in methanogenic archaea. A crystal structure of methanogenic-type SerRS revealed a motif within the active site (serine ordering loop; SOL), which undergoes a notable induced-fit rearrangement during serine binding. The loop rearranges from a disordered conformation in the unliganded enzyme, to an ordered structure comprising an α-helix followed by a loop. We performed kinetic and thermodynamic analyses of SerRS variants to establish the role of the SOL in serylation. Thermodynamic data confirmed a linkage between binding of serine and α-helix formation, previously described by the crystallographic analysis. The ability of the SOL to adopt the observed secondary structure was recognized as essential for serine activation. Mutation of Gln400, which according to the structural data establishes the main connection between the serine and the SOL, produced only modest kinetic effects. Kinetic data offer new insights into the coupling of the conformational change with active site assembly. Productive positioning of the SOL may be driven by the interaction between Trp396 and the serine α-amino group. Rapid kinetics reveals that His250, a non-SOL residue, is essential for transfer of serine to tRNA. Modeling data established that accommodation of the tRNA within the active site may require movement of the SOL. This would enable His250 to assist in productive positioning of the 3′-end of the tRNA for the aminoacyl transfer. Thus, the rearrangements of the SOL conformationally adjust the active site for both reaction steps.     

Complete genome sequence of a nonculturable Methanococcus maripaludis strain extracted in a metagenomic survey of petroleum reservoir fluids

Extraction of genome sequences from metagenomic data is crucial for reconstructing the metabolism of microbial communities that cannot be mimicked in the laboratory. A complete Methanococcus maripaludis genome was generated from metagenomic data derived from a thermophilic subsurface oil reservoir. M. maripaludis is a hydrogenotrophic methanogenic species that is common in mesophilic saline environments. Comparison of the genome from the thermophilic, subsurface environment with the genome of the type species will provide insight into the adaptation of a methanogenic genome to an oil reservoir environment.     

Lysine-2,3-aminomutase and beta-lysine acetyltransferase genes of methanogenic archaea are salt induced and are essential for the biosynthesis of Nepsilon-acetyl-beta-lysine and growth at high salinity

The compatible solute N(epsilon)-acetyl-beta-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of N(epsilon)-acetyl-beta-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and beta-lysine acetyltransferase (ablB), which are assumed to catalyze N(epsilon)-acetyl-beta-lysine formation from alpha-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei G?1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei G?1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Delta(abl) mutants of M. maripaludis no longer produced N(epsilon)-acetyl-beta-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for N(epsilon)-acetyl-beta-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.     

Dual mode recognition of two isoacceptor tRNAs by mammalian mitochondrial seryl-tRNA synthetase

Animal mitochondrial translation systems contain two serine tRNAs, corresponding to the codons AGY (Y = U and C) and UCN (N = U, C, A, and G), each possessing an unusual secondary structure; tRNA(GCU)(Ser) (for AGY) lacks the entire D arm, whereas tRNA(UGA)(Ser) (for UCN) has an unusual cloverleaf configuration. We previously demonstrated that a single bovine mitochondrial seryl-tRNA synthetase (mt SerRS) recognizes these topologically distinct isoacceptors having no common sequence or structure. Recombinant mt SerRS clearly footprinted at the TPsiC loop of each isoacceptor, and kinetic studies revealed that mt SerRS specifically recognized the TPsiC loop sequence in each isoacceptor. However, in the case of tRNA(UGA)(Ser), TPsiC loop-D loop interaction was further required for recognition, suggesting that mt SerRS recognizes the two substrates by distinct mechanisms. mt SerRS could slightly but significantly misacylate mitochondrial tRNA(Gln), which has the same TPsiC loop sequence as tRNA(UGA)(Ser), implying that the fidelity of mitochondrial translation is maintained by kinetic discrimination of tRNAs in the network of aminoacyl-tRNA synthetases.     

Methanocaldococcus jannaschii prolyl-tRNA synthetase charges tRNA(Pro) with cysteine

Methanocaldococcus jannaschii prolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and S?ll, D. (2000) Science 287, 479-482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000) Biochemistry 39, 7792-7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinant M. jannaschii ProRS misaminoacylates M. jannaschii tRNA(Pro) with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschii tRNA(Cys) with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludis cysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNA(Pro) readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNA(Cys) in M. jannaschii.     

Inactivation of the selB gene in Methanococcus maripaludis: effect on synthesis of selenoproteins and their sulfur-containing homologs

The genome of Methanococcus maripaludis harbors genes for at least six selenocysteine-containing proteins and also for homologs that contain a cysteine codon in the position of the UGA selenocysteine codon. To investigate the synthesis and function of both the Se and the S forms, a mutant with an inactivated selB gene was constructed and analyzed. The mutant was unable to synthesize any of the selenoproteins, thus proving that the gene product is the archaeal translation factor (aSelB) specialized for selenocysteine insertion. The wild-type form of M. maripaludis repressed the synthesis of the S forms of selenoproteins, i.e., the selenium-independent alternative system, in selenium-enriched medium, but the mutant did not. We concluded that free selenium is not involved in regulation but rather a successional compound such as selenocysteyl-tRNA or some selenoprotein. Apart from the S forms, several enzymes from the general methanogenic route were affected by selenium supplementation of the wild type or by the selB mutation. Although the growth of M. maripaludis on H(2)/CO(2) is only marginally affected by the selB lesion, the gene is indispensable for growth on formate because M. maripaludis possesses only a selenocysteine-containing formate dehydrogenase.     

Bacterial and eukaryotic systems collide in the three Rs of Methanococcus

Methanococcus maripaludis S2 is a methanogenic archaeon with a well-developed genetic system. Its mesophilic nature offers a simple system in which to perform complementation using bacterial and eukaryotic genes. Although information-processing systems in archaea are generally more similar to those in eukaryotes than those in bacteria, the order Methanococcales has a unique complement of DNA replication proteins, with multiple MCM (minichromosome maintenance) proteins and no obvious originbinding protein. A search for homologues of recombination and repair proteins in M. maripaludis has revealed a mixture of bacterial, eukaryotic and some archaeal-specific homologues. Some repair pathways appear to be completely absent, but it is possible that archaeal-specific proteins could carry out these functions. The replication, recombination and repair systems in M. maripaludis are an interesting mixture of eukaryotic and bacterial homologues and could provide a system for uncovering novel interactions between proteins from different domains of life.     

Yeast seryl tRNA synthetase: two sets of substrate sites involved in aminoacylation.

Seryl tRNA synthetase from Saccharomyces Carlsbergensis C836 contains two sets of sites for tRNASer, L-serine, and Mg2+-ATP, both of which are involved in aminoacylation. This is based on the following experimental results: (a) at low serine concentrations, second order kinetics in tRNASer are observed; (b) biphasic kinetics result when the amino acid is the varied substrate indicating anticooperative binding of two serine molecules to the synthetase; (c) when two molecules of serine are bound the rate of aminoacylation increases strongly and becomes first order in tRNASer; (d) the involvement of more than one site for Mg2+ and ATP is deduced from systematic variations of the concentrations of Mg2+ and ATP. Implications of the anticooperative binding of the substrates for possible reaction mechanisms are discussed. The results indicate that under normal conditions, the activity of seryl tRNA synthetase is regulated mainly by tRNASer while at high serine concentrations regulation by the amino acid itself prevails.     

The long extra arms of human tRNA((Ser)Sec) and tRNA(Ser) function as major identify elements for serylation in an orientation-dependent, but not sequence-specific manner.

Selenocysteine tRNA [tRNA((Ser)Sec)] is charged with serine by the same seryl-tRNA synthetase (SerRS) as the canonical serine tRNAs. Using site-directed mutagenesis, we have introduced a series of mutations into human tRNA((Ser)Sec) and tRNA(Ser) in order to study the identity elements of tRNA((Ser)Sec) for serylation and the effect of the orientation of the extra arm. Our results show that the long extra arm is one of the major identity elements for both tRNA(Ser) and tRNA((Ser)Sec) and gel retardation assays reveal that it appears to be a prerequisite for binding to the cognate synthetase. The long extra arm functions in an orientation-dependent, but not in a sequence-specific manner. The discriminator base G73 is another important identity element of tRNA((Ser)Sec), whereas the T- and D-arms play a minor role for the serylation efficiency.