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PubMed2003

Arabidopsis proteins containing similarity to the universal stress protein domain of bacteria.

We have collected a set of 44 Arabidopsis proteins with similarity to the USPA (universal stress protein A of Escherichia coli) domain of bacteria. The USPA domain is found either in small proteins, or it makes up the N-terminal portion of a larger protein, usually a protein kinase. Phylogenetic tree analysis based upon a multiple sequence alignment of the USPA domains shows that these domains of protein kinases 1.3.1 and 1.3.2 form distinct groups, as do the protein kinases 1.4.1. This indicates that their USPA domain structures have diverged appreciably and suggests that they may subserve distinct cellular functions. Two USPA fold classes have been proposed: one based on Methanococcus jannaschii MJ0577 (1MJH) that binds ATP, and the other based on the Haemophilus influenzae universal stress protein (1JMV), highly similar to E. coli UspA, which does not bind ATP. A set of common residues involved in ATP binding in 1MJH and conserved in similar bacterial sequences is also found in a distinct cluster of Arabidopsis sequences. Threading analysis, which examines aspects of secondary and tertiary structure, confirms this Arabidopsis sequence cluster as highly similar to 1MJH. This structural approach can distinguish between the characteristic fold differences of 1MJH-like and 1JMV-like bacterial proteins and was used to assign the complete set of candidate Arabidopsis proteins to one of these fold classes. It is clear that all the plant sequences have arisen from a 1MJH-like ancestor.

Adenosine Triphosphate↗

PubMed2006

Structure of the ribosomal protein L1-mRNA complex at 2.1 A resolution: common features of crystal packing of L1-RNA complexes.

The crystal structure of a hybrid complex between the bacterial ribosomal protein L1 from Thermus thermophilus and a Methanococcus vannielii mRNA fragment containing an L1-binding site was determined at 2.1 A resolution. It was found that all polar atoms involved in conserved protein-RNA hydrogen bonds have high values of density in the electron-density map and that their hydrogen-bonding capacity is fully realised through interactions with protein atoms, water molecules and K(+) ions. Intermolecular contacts were thoroughly analyzed in the present crystals and in crystals of previously determined L1-RNA complexes. It was shown that extension of the RNA helices providing canonical helix stacking between open-open or open-closed ends of RNA fragments is a common feature of these and all known crystals of complexes between ribosomal proteins and RNAs. In addition, the overwhelming majority of complexes between ribosomal proteins and RNA molecules display crystal contacts formed by the central parts of the RNA fragments. These contacts are often very extensive and strong and it is proposed that they are formed in the saturated solution prior to crystal formation.

Amino Acid Sequence↗

PubMed2006

The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase.

The activation of the beta-carboxyl group of aspartate catalyzed by aspartokinase is the commitment step to amino-acid biosynthesis in the aspartate pathway. The first structure of a microbial aspartokinase, that from Methanococcus jannaschii, has been determined in the presence of the amino-acid substrate L-aspartic acid and the nucleotide product MgADP. The enzyme assembles into a dimer of dimers, with the interfaces mediated by both the N- and C-terminal domains. The active-site functional groups responsible for substrate binding and specificity have been identified and roles have been proposed for putative catalytic functional groups.

Archaeal Proteins↗

PubMed2004

Direct demonstration of carbamoyl phosphate formation on the C-terminal domain of carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase synchronizes the utilization of two ATP molecules at duplicated ATP-grasp folds to catalyze carbamoyl phosphate formation. To define the dedicated functional role played by each of the two ATP sites, we have carried out pulse/labeling studies using the synthetases from Aquifex aeolicus and Methanococcus jannaschii, hyperthermophilic organisms that encode the two ATP-grasp folds on separate subunits. These studies allowed us to differentially label each active site with [gamma-(32)P]ATP and determine the fate of the labeled gamma-phosphate in the synthetase reaction. Our results provide the first direct demonstration that enzyme-catalyzed transfer of phosphate from ATP to carbamate occurs on the more C-terminal of the two ATP-grasp folds. These findings rule out one mechanism proposed for carbamoyl phosphate synthetase, where one ATP acts as a molecular switch, and provide additional support for a sequential reaction mechanism where the gamma-phosphate groups of both ATP molecules are transferred to reactants. CP synthesis by subunit C in our single turnover pulse/chase assays did not require subunit N, but subunit N was required for detectable CP synthesis in the traditional continuous assay. These findings suggest that cross-talk between domain N and C is required for product release from subunit C.

Adenosine Triphosphate↗

PubMed2005

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine.

The Methanococcus jannaschii tRNA(Tyr)/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-L-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 A, respectively, for comparison with the published structure of TyrRS complexed with tRNA(Tyr) and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257-263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through pi-stacking and hydrogen bonding interactions. Loop 133-143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNA(Tyr). Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133-143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over L-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids.

Crystallography, X-Ray↗

PubMed2001

Unusual evolutionary history of the tRNA splicing endonuclease EndA: relationship to the LAGLIDADG and PD-(D/E)XK deoxyribonucleases.

The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.

Binding Sites↗

PubMed2004

Transcriptional fidelity and proofreading in Archaea and implications for the mechanism of TFS-induced RNA cleavage.

We have addressed the question whether TFS, a protein that stimulates the intrinsic cleavage activity of the archaeal RNA polymerase, is able to improve the fidelity of transcription in Methanococcus. Using non-specific transcription experiments, we could demonstrate that misincorporation of non-templated nucleotides is reduced in the presence of TFS. A more detailed analysis revealed that elongation complexes containing a misincorporated nucleotide were arrested, but could be reactivated by TFS. RNase as well as exonuclease III footprinting experiments demonstrated that this arrest was not combined with extended backtracking. Analysis of paused elongation complexes demonstrated that TFS is able to induce a cleavage resynthesis cycle in such complexes, which resulted in the accumulation of dinucleotides corresponding to the last two nucleotides of the transcript. Further analysis of cleavage products revealed that, even under conditions that strongly promote misincorporation, still 50% of the released dinucleotides were correctly incorporated. Therefore, we assume that pausing of elongation complexes is an important determinant of TFS-induced RNA cleavage from the 3' end. As the incorporation rate of wrong nucleotides is about 700-fold reduced, it is possible that this delay also provides an appropriate time window for cleavage induction in order to maintain transcriptional fidelity by preventing misincorporation.

Archaea↗

PubMed2005

Population density-dependent regulation of exopolysaccharide formation in the hyperthermophilic bacterium Thermotoga maritima.

Co-cultivation of the hyperthermophiles Thermotoga maritima and Methanococcus jannaschii resulted in fivefold higher T. maritima cell densities when compared with monoculture as well as concomitant formation of exopolysaccharide and flocculation of heterotroph-methanogen cellular aggregates. Transcriptional analysis of T. maritima cells from these aggregates using a whole genome cDNA microarray revealed the induction of a putative exopolysaccharide synthesis pathway, regulated by intracellular levels of cyclic diguanosine 3',5'-(cyclic)phosphate (cyclic di-GMP) and mediated by the action of several GGDEF proteins, including a putative diguanylate cyclase (TM1163) and a putative phosphodiesterase (TM1184). Transcriptional analysis also showed that TM0504, which encodes a polypeptide containing a motif common to known peptide-signalling molecules in mesophilic bacteria, was strongly upregulated in the co-culture. Indeed, when a synthetically produced peptide based on TM0504 was dosed into the culture at ecologically relevant levels, the production of exopolysaccharide was induced at significantly lower cell densities than was observed in cultures lacking added peptide. In addition to identifying a pathway for polysaccharide formation in T. maritima, these results point to the existence of peptide-based quorum sensing in hyperthermophilic bacteria and indicate that cellular communication should be considered as a component of the microbial ecology within hydrothermal habitats.

Bacterial Proteins↗

PubMed2004

The unusual methanogenic seryl-tRNA synthetase recognizes tRNASer species from all three kingdoms of life.

The methanogenic archaea Methanococcus jannaschii and M. maripaludis contain an atypical seryl-tRNA synthetase (SerRS), which recognizes eukaryotic and bacterial tRNAsSer, in addition to the homologous tRNASer and tRNASec species. The relative flexibility in tRNA recognition displayed by methanogenic SerRSs, shown by aminoacylation and gel mobility shift assays, indicates the conservation of some serine determinants in all three domains. The complex of M. maripaludis SerRS with the homologues tRNASer was isolated by gel filtration chromatography. Complex formation strongly depends on the conformation of tRNA. Therefore, the renaturation conditions for in vitro transcribed tRNASer(GCU) isoacceptor were studied carefully. This tRNA, unlike many other tRNAs, is prone to dimerization, possibly due to several stretches of complementary oligonucleotides within its sequence. Dimerization is facilitated by increased tRNA concentration and can be diminished by fast renaturation in the presence of 5 mm magnesium chloride.

Anticodon↗

PubMed1998

A tRNA(Glu) gene from the hyperthermophilic archaeon Pyrococcus furiosus contains the 3'-terminal CCA sequence of the mature tRNA.

We cloned a gene encoding tRNA(Glu) of the hyperthermophilic archaeon Pyrococcus furiosus. This gene contains the CCA sequence corresponding to the 3'-terminus of the mature tRNA. It is known that, like in eukaryal tRNAs, the CCA-termini of archaeal tRNAs are generally not encoded. Therefore, we analyzed all tRNA genes in the genome of Methanococcus jannaschii estimated by its whole genome sequence. Twenty-one of 37 listed tRNA genes contained the 3'-terminal CCA sequence. The corresponding M. jannaschii tRNA(Glu) gene does not contain the CCA sequence, although the tRNA sequences of the M. jannaschii and P. furiosus tRNA(Glu) genes are 86% identical.

Anticodon↗

PubMed2001

Similar subunit architecture of archaeal and eukaryal RNA polymerases.

Protein interactions among RNA polymerase small subunits from the archaeon Methanococcus jannaschii were investigated using affinity pulldown assays in pairwise and higher-order combinations. In the most extensive study of archaeal RNA polymerase subunit interactions to date, including 37 pairs of proteins, 10 ternary combinations, and three quaternary combinations, we found evidence for pairwise interactions of subunit D with subunits L and N, and a ternary complex of subunits D, L and N. No other small subunit interactions occurred. These results are consistent with interactions observed in a crystal structure of eukaryotic RNA polymerase II and support a common archaeal/eukaryal RNA polymerase architecture. We further propose that subunit E" is not an integral member of archaeal RNA polymerases. Finally, we discuss the relative accuracy of the various methods that have been used to predict protein-protein interactions in RNA polymerase.

Blotting, Western↗

PubMed2002

Molecular characterization of phosphoglycerate mutase in archaea.

The interconversion of 3-phosphoglycerate and 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). In bacteria and eukaryotes two structurally distinct enzymes have been found, a cofactor-dependent and a cofactor-independent (iPGM) type. Sequence analysis of archaeal genomes did not find PGMs of either kind, but identified a new family of proteins, distantly related to iPGMs. In this study, these predicted archaeal PGMs from Pyrococcus furiosus and Methanococcus jannaschii have been functionally produced in Escherichia coli, and characterization of the purified proteins has confirmed that they are iPGMs. Analysis of the available microbial genomes indicates that this new type of iPGM is widely distributed among archaea and also encoded in several bacteria. In addition, as has been demonstrated in certain bacteria, some archaea appear to possess an alternative, cofactor-dependent PGM.

Amino Acid Sequence↗

PubMed1998

The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait.

Inspection of the genomes for the bacteria Bacillus subtilis 168, Borrelia burgdorferi B31, Escherichia coli K-12, Haemophilus influenzae KW20, Helicobacter pylori 26695, Mycoplasma genitalium G-37, and Synechocystis sp PCC 6803 and for the archaeons Archaeoglobus fulgidus VC-16 DSM4304, Methanobacterium thermoautotrophicum delta H, and Methanococcus jannaschii DSM2661 revealed that each contains at least one ORF whose predicted product displays sequence features characteristic of eukaryote-like protein-serine/threonine/tyrosine kinases and protein-serine/threonine/tyrosine phosphatases. Orthologs for all four major protein phosphatase families (PPP, PPM, conventional PTP, and low molecular weight PTP) were present in the bacteria surveyed, but not all strains contained all types. The three archaeons surveyed lacked recognizable homologs of the PPM family of eukaryotic protein-serine/threonine phosphatases; and only two prokaryotes were found to contain ORFs for potential phosphatases from all four major families. Intriguingly, our searches revealed a potential ancestral link between the catalytic subunits of microbial arsenate reductases and the protein-tyrosine phosphatases; they share similar ligands (arsenate versus phosphate) and features of their catalytic mechanism (formation of arseno-versus phospho-cysteinyl intermediates). It appears that all prokaryotic organisms, at one time, contained the genetic information necessary to construct protein phosphorylation-dephosphorylation networks that target serine, threonine, and/or tyrosine residues on proteins. However, the potential for functional redundancy among the four protein phosphatase families has led many prokaryotic organisms to discard one, two, or three of the four.

Amino Acid Sequence↗

PubMed1999

Detecting alien genes in bacterial genomes.

We present new methods for calculating codon bias of a group of genes or an individual gene relative to a standard gene class. This method is suitable for identifying alien (e.g., horizontally transferred) and highly expressed genes. In yeast and several bacterial genomes, highly expressed genes typically include ribosomal protein genes, elongation factors, chaperonins (heat shock proteins), and a subset of genes involved in glycolysis generally essential in exponential growth. Highly expressed genes of the Synechocystis genome feature several photosystem II genes, and highly expressed genes in several methanogens (Methanococcus jannaschii, M. thermoautotrophicum) are essential for methanogenesis. Alien genes mostly consist of ORFs of unknown function, transposases, prophage genes, and restriction/modification enzymes. Notably, nuclear ribosomal proteins of yeast are highly expressed, whereas mitochondrial ribosomal protein genes appear to be alien genes. Alien genes often occur in clusters, suggesting in these cases that transfer events entail several genes.

Borrelia burgdorferi Group↗

PubMed2005

RNA-dependent cysteine biosynthesis in archaea.

Several methanogenic archaea lack cysteinyl-transfer RNA (tRNA) synthetase (CysRS), the essential enzyme that provides Cys-tRNA(Cys) for translation in most organisms. Partial purification of the corresponding activity from Methanocaldococcus jannaschii indicated that tRNA(Cys) becomes acylated with O-phosphoserine (Sep) but not with cysteine. Further analyses identified a class II-type O-phosphoseryl-tRNA synthetase (SepRS) and Sep-tRNA:Cys-tRNA synthase (SepCysS). SepRS specifically forms Sep-tRNA(Cys), which is then converted to Cys-tRNA(Cys) by SepCysS. Comparative genomic analyses suggest that this pathway, encoded in all organisms lacking CysRS, can also act as the sole route for cysteine biosynthesis. This was proven for Methanococcus maripaludis, where deletion of the SepRS-encoding gene resulted in cysteine auxotrophy. As the conversions of Sep-tRNA to Cys-tRNA or to selenocysteinyl-tRNA are chemically analogous, the catalytic activity of SepCysS provides a means by which both cysteine and selenocysteine may have originally been added to the genetic code.

Adenosine Triphosphate↗

PubMed1998

Crystal structure and evolution of a transfer RNA splicing enzyme.

The splicing of transfer RNA precursors is similar in Eucarya and Archaea. In both kingdoms an endonuclease recognizes the splice sites and releases the intron, but the mechanism of splice site recognition is different in each kingdom. The crystal structure of the endonuclease from the archaeon Methanococcus jannaschii was determined to a resolution of 2.3 angstroms. The structure indicates that the cleavage reaction is similar to that of ribonuclease A and the arrangement of the active sites is conserved between the archaeal and eucaryal enzymes. These results suggest an evolutionary pathway for splice site recognition.

Amino Acid Sequence↗

PubMed2000

One polypeptide with two aminoacyl-tRNA synthetase activities.

The genome sequences of certain archaea do not contain recognizable cysteinyl-transfer RNA (tRNA) synthetases, which are essential for messenger RNA-encoded protein synthesis. However, a single cysteinyl-tRNA synthetase activity was detected and purified from one such organism, Methanococcus jannaschii. The amino-terminal sequence of this protein corresponded to the predicted sequence of prolyl-tRNA synthetase. Biochemical and genetic analyses indicated that this archaeal form of prolyl-tRNA synthetase can synthesize both cysteinyl-tRNA(Cys) and prolyl-tRNA(Pro). The ability of one enzyme to provide two aminoacyl-tRNAs for protein synthesis raises questions about concepts of substrate specificity in protein synthesis and may provide insights into the evolutionary origins of this process.

Amino Acyl-tRNA Synthetases↗

PubMed1998

Mercury methylation by interspecies hydrogen and acetate transfer between sulfidogens and methanogens.

Cocultures of Desulfovibrio desulfuricans and Methanococcus maripaludis grew on sulfate-free lactate medium while vigorously methylating Hg2+. Individually, neither bacterium could grow or methylate mercury in this medium. Similar synergistic growth of sulfidogens and methanogens may create favorable conditions for Hg2+ methylation in low-sulfate anoxic freshwater sediments.

Acetic Acid↗