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PubMed1995

Identification of a eukaryotic-like protein kinase gene in Archaebacteria.

Primary sequence patterns based on known conserved sites in eukaryotic protein kinases were used to search for eukaryotic-like protein kinase sequences in a six-frame translation of the bacterial subsection of GenBank. This search identified a previously unrecognized eukaryotic-like protein kinase gene in three related methanogenic archaebacteria, Methanococcus vannielii, M. voltae, and M. thermolithotrophicus. The proposed coding sequences are located in orthologous open reading frames (ORFs): ORF547, ORF294, and ORF114, respectively. The C-terminus of the ORFs contains 9 of the 11 subdomains characteristically conserved within the eukaryotic protein kinase catalytic domain. The N-terminus of the ORFs is similar to a putative glycoprotease in Pasteurella haemolytica and its homologue in Escherichia coli, the orfX gene. This is the first report of a eukaryotic-like protein kinase sequence observed in Archaebacteria.

Amino Acid Sequence↗
PubMed1997

Cloning, expression, and crystallization of a hyperthermophilic protein that is homologous to the eukaryotic translation initiation factor, eIF5A.

A gene coding for a protein homologous to a translation initiation factor of eukaryotes, eIF5A, was cloned from Methanococcus jannaschii, a hyperthermophile with an optimum growth temperature of 85 degrees C. The protein was overexpressed, purified and crystallized. The crystals were obtained by vapor diffusion method with 8% PEG 4000 as precipitant and belong to space group P4(1)22 with unit cell dimensions a = b = 45.52 A and c = 155.59 A. These crystals diffract to at least 2.2 A resolution.

Cloning, Molecular↗
PubMed2005

Universal biases in protein composition of model prokaryotes.

The levels of cellular organization in living organisms are the results of a variety of selection pressures. We have investigated here the final outcome of this integrated selective process in proteins of the best known microbial models Escherichia coli, Bacillus subtilis, and Methanococcus jannaschii, supposed to have undergone separate evolution for more than 1 billion years. Using multivariate analysis methods, including correspondence analysis, we studied the overall amino acid composition of all proteins making a proteome. Starting from and further developing previous results that had pointed out some general forces driving the amino acid composition of the proteomes of these model bacteria, we explored the correlations existing between the structure and functions of the proteins forming a proteome and their amino acid composition. The electric charge of amino acids measured against hydrophobicity creates a highly homogeneous cluster, made exclusively of proteins that are core components of the cytoplasmic membrane of the cell (integral inner membrane proteins). A second bias is imposed by the G+C content of the genome, indicating that protein functions are so robust with respect to amino acid changes that they can accommodate a large shift in the nucleotide content of the genome. A remarkable role of aromatic amino acids was uncovered. Expressed orphan proteins are enriched in these residues, suggesting that they might participate in a process of gain of function during evolution.

Amino Acids↗
PubMed2007

Relative tolerance of mesostable and thermostable protein homologs to extensive mutation.

Evolvability, designability, and plasticity of a protein are properties that are important to protein engineers, but difficult to quantify. Here, we directly compare homologous AroQ chorismate mutases from the thermophile Methanococcus jannaschii and the mesophile Escherichia coli with respect to their capacity to accommodate extensive mutation. The N-terminal helix comprising about 40% of these proteins was randomized at the genetic level using a binary pattern of hydrophobic and hydrophilic residues based on the respective wild-type sequences. Catalytically active library members were identified by a survival-selection assay in a chorismate mutase-deficient E. coli strain. Functional variants were found approximately approximately 10-times more frequently with the thermostable protein compared to its mesostable counterpart. Moreover, detailed sequence analysis revealed that functional M. jannaschii enzyme variants contained a smaller number of conserved residues and tolerated greater variability at individual sequence positions. Our results thus highlight the greater robustness of the thermostable protein with respect to amino acid substitution, while identifying specific sites important for constructing active enzymes. Overall, they support the notion that redesign projects will benefit from using a thermostable starting structure, even at very high mutational loads.

Amino Acid Sequence↗
PubMed1994

Expressed sequence tags identify a human isolog of the suil translation initiation factor.

The complete cDNA sequence of a human isolog of the yeast suil translation initiation factor gene was obtained by assembling over 40 expressed sequence tags (ESTs) for this gene obtained from a variety of tissue-specific cDNA libraries. The human suilisol gene product is a 113 amino-acid polypeptide similar to proteins known from yeast, rice, mosquito, and Methanococcus. The identification of suilisol illustrates the utility of assemblies of independent ESTs for deriving full-length cDNA sequences for new human genes.

Amino Acid Sequence↗
PubMed1997

The stabilizing residues and the functional domains in the hyperthermophilic V-ATPase of Desulfurococcus.

To clarify a universal mechanism of the intramolecular rotation of ATP-synthase, an operon encoding a stable, ancestral ATPase was cloned from a heterotrophic archaeum Desulfurococcus strain SY. The operon of about 7 kbp contained genes E, C, G, A, B and D encoding subunits with predicted molecular weights of 23,217, 41,659, 11,499, 65,476, 52,295, and 24,897, respectively. The sequence was compared with that of Na-ATPase of Enterococcus hirae, A-ATPase of Halobacterium salinarium, V-ATPase of Methanosarcina mazei, and ATP synthase of Methanococcus jannaschii, which are homologous. (1) The cause of hyperthermostability: The main exchanges in the amino acid residues of hyperthermophilic proteins included Asp --> Glu (11 residues of A subunit of E.h.) and, Ser --> Ala. (2) The domains needed for the intramolecular rotation: The domains similar to those established in F-type ATPases were also found in the V-type ATPases of species with a different energy metabolism.

Adenosine Triphosphatases↗
PubMed1997

Cloning and sequencing of aspartate aminotransferase from Thermus aquaticus YT1.

A 39-base oligonucleotide "guessmer" probe, based on partial N-terminal sequence analysis of the aspartate aminotransferase purified from Thermus aquaticus strain YT1, was used to screen a genomic library prepared from T. aquaticus DNA. A 1842 bp DNA fragment was isolated that proved to contain the coding sequence for the aspartate aminotransferase. The gene is 1152 bases long and codes for a protein of 383 amino acid residues. The amino acid sequence obtained showed 88.7%, 45.1% and 32.9% identity of sequence with those of thermostable aspartate aminotransferases from T. thermophilus, Bacillus YM2, and Sulfolobus solfataricus, respectively. It showed 39.1% identity with one of the gene products tentatively identified as aspartate aminotransferase from the methanogenic archaebacterium Methanococcus jannaschii. Neither the amino acid compositions nor the aligned amino acid sequences provides any obvious clue as to the origin of thermal stability in this group of enzymes.

Amino Acid Sequence↗
PubMed1999

RNA polymerase subunit H features a beta-ribbon motif within a novel fold that is present in archaea and eukaryotes.

The archaeal H and eukaryotic RPB5 RNA polymerase subunits are highly homologous and are likely to play a fundamental role in transcription that extends from archaea to humans. We report the structure of subunit H, in solution, from the archaeon Methanococcus jannaschii using multidimensional nuclear magnetic resonance. The structure reveals a novel fold containing a four-stranded mixed beta sheet that is flanked on one side by three short helices. The dominant feature is beta-ribbon motif, which presents a hydrophobic, basic surface, and defines a general RNA polymerase architectural scaffold.

Amino Acid Sequence↗
PubMed1999

Structural compensation in an archaeal selenocysteine transfer RNA.

A new type of structural compensation between the lengths of two perpendicularly oriented RNA double helices was found in the archaeal selenocysteine tRNA from Methanococcus jannascii. This tRNA contains only four base-pairs in the T-stem, one base-pair less than in all other cytosolic tRNAs. Our analysis shows that such a T-stem in an otherwise normal tRNA cannot guarantee the formation of the normal interactions between the D and T-loops. The absence of these interactions would affect the juxtaposition of the two tRNA helical domains, potentially damaging the tRNA function. In addition to the short T-stem, this tRNA possesses another unprecedented feature, a very long D-stem consisting of seven base-pairs. Taken as such, a seven base-pair D-stem will also disrupt the normal interaction between the D and T-loops. On the other hand, the presence of the universal nucleotides in both the D and T-loops suggests that these loops probably interact with each other in the same way as in other tRNAs. Here, we demonstrate that the short T-stem and the long D-stem can naturally compensate each other, thus providing the normal D/T interactions. Molecular modeling has helped suggest a detailed scheme of mutual compensation between these two unique structural aspects of the archaeal selenocysteine tRNA. In the light of this analysis, other structural and functional characteristics of the selenocysteine tRNAs are discussed.

Base Sequence↗
PubMed2000

Small heat-shock protein structures reveal a continuum from symmetric to variable assemblies.

The small heat-shock proteins (sHSPs) form a diverse family of proteins that are produced in all organisms. They function as chaperone-like proteins in that they bind unfolded polypeptides and prevent uncontrolled protein aggregation. Here, we present parallel cryo-electron microscopy studies of five different sHSP assemblies: Methanococcus jannaschii HSP16.5, human alphaB-crystallin, human HSP27, bovine native alpha-crystallin, and the complex of alphaB-crystallin and unfolded alpha-lactalbumin. Gel-filtration chromatography indicated that HSP16.5 is the most monodisperse, while HSP27 and the alpha-crystallin assemblies are more polydisperse. Particle images revealed a similar trend showing mostly regular and symmetric assemblies for HSP16.5 particles and the most irregular assemblies with a wide range of diameters for HSP27. A symmetry test on the particle images indicated stronger octahedral symmetry for HSP16.5 than for HSP27 or the alpha-crystallin assemblies. A single particle reconstruction of HSP16.5, based on 5772 particle images with imposed octahedral symmetry, resulted in a structure that closely matched the crystal structure. In addition, the cryo-EM reconstruction revealed internal density presumably corresponding to the flexible 32 N-terminal residues that were not observed in the crystal structure. The N termini were found to partially fill the central cavity making it unlikely that HSP16.5 sequesters denatured proteins in the cavity. A reconstruction calculated without imposed symmetry confirmed the presence of at least loose octahedral symmetry for HSP16.5 in contrast to the other sHSPs examined, which displayed no clear overall symmetry. Asymmetric reconstructions for the alpha-crystallin assemblies, with an additional mass selection step during image processing, resulted in lower resolution structures. We interpret the alpha-crystallin reconstructions to be average representations of variable assemblies and suggest that the resolutions achieved indicate the degree of variability. Quaternary structural information derived from cryo-electron microscopy is related to recent EPR studies of the alpha-crystallin domain fold and dimer interface of alphaA-crystallin.

Animals↗
PubMed2000

Crystal structure of a dimeric archaeal splicing endonuclease.

The splicing endonuclease from Archaeoglobus fulgidus (AF) belongs to the homodimeric family of splicing endonucleases, thought to have evolved from the homotetrameric endonucleases. We report here the crystal structure of the AF endonuclease determined at 2.8 A. The crystal structure of the full-length AF endonuclease contains a homodimer, with each monomer consisting of two homologous repeats joined together by an extended polypeptide chain of ten amino acid residues. The C-terminal repeat has a strong homology to that of a single subunit of the previously determined homotetrameric tRNA splicing endonuclease from Methanococcus jannaschii (MJ), indicating its role in catalysis. The N-terminal repeat is a more degenerate form of the MJ enzyme. Thus the N-terminal repeat is a "non-active" endonuclease fold evolved from the "active" one. By detailed comparison of the structures of the N-terminal and the C-terminal repeats, the binding region for RNA substrates containing a bulge-helix-bulge motif can be identified. Based on the identified RNA-binding region, a cation-pi interaction is suggested to be responsible for coordinating activities between the two active sites. In addition, the full-length AF endonuclease can adopt a higher-ordered fibrous structure in solution, as revealed by the unusual crystallographic packing interactions and other biochemical analysis. This 4(3)-fold fibrous structure adopted by the full-length enzyme is inaccessible to the RNA substrate and is largely stabilized by the first 60 amino acid residues. A mutated form of AF endonuclease with its first 60 residues removed catalyzes the cleavage reaction at a significantly higher rate. Whether there is any role in vivo for this structure-mediated modulation of activity remains to be determined.

Amino Acid Sequence↗
PubMed2002

Prevention of mis-aminoacylation of a dual-specificity aminoacyl-tRNA synthetase.

Accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetase is essential for the fidelity of protein synthesis. For Methanococcus jannaschii tRNA(Pro), accuracy is difficult because the cognate prolyl-tRNA synthetase also recognizes and aminoacylates tRNA(Cys) with cysteine. We show here that the unmodified transcript of M. jannaschii tRNA(Pro) is indeed mis-acylated with cysteine. However, the origin of mis-charging is not at the anticodon or acceptor stem, the two hotspots for tRNA(Pro) and tRNA(Cys) identity determinants. Instead, replacement of the D loop in the tRNA core with that of tRNA(Cys) suppresses mis-charging with cysteine without compromising the activity of aminoacylation with proline. The reduced level of cysteine activity of the chimera is not due an editing response of the synthetase and is consistent with a relaxed sensitivity of the tRNA to the analog thiaproline in aminoacylation with cysteine. We suggest that mis-acylation is not due to the presence of cysteine determinants, but to a mis-placed 3' end into the cysteine catalytic site that activates and transfers cysteine to the tRNA. Prevention of mis-placement by alteration of the core structure or by nucleotide modifications in the tRNA illustrates a novel strategy of the dual-specificity synthetase.

Acylation↗
PubMed2002

Amino acid activation of a dual-specificity tRNA synthetase is independent of tRNA.

Transfer RNA can play a role in amino acid activation by aminoacyl-tRNA synthetases. For the prolyl-tRNA synthetase (ProRS) of Methanococcus jannaschii, which activates both proline and cysteine, the role of tRNA in amino acid selection and activation is of interest in the effort to understand the mechanism of the dual-specificity. While activation of proline does not require tRNA, whether or not tRNA is required in the activation of cysteine has been a matter of debate. Here, investigation of a series of buffer conditions shows that activation of cysteine occurs without tRNA in a wide-range of buffers. However, the extent of cysteine activation is strongly buffer-dependent, varying over a 180-fold range. In contrast, the extent of proline activation is much less sensitive to buffer conditions, varying over only a 36-fold range. We also find that addition of tRNA has a small threefold stimulatory effect on cysteine activation. The lack of a major role of tRNA in activation of cysteine suggests that the dual-specificity enzyme must distinguish cysteine from proline directly, without the assistance of each cognate tRNA, to achieve the necessary specificity required for protein synthesis.

Amino Acyl-tRNA Synthetases↗
PubMed2002

Comparative genomics using data mining tools.

We have analysed the genomes of representatives of three kingdoms of life, namely, archaea, eubacteria and eukaryota using data mining tools based on compositional analyses of the protein sequences. The representatives chosen in this analysis were Methanococcus jannaschii, Haemophilus influenzae and Saccharomyces cerevisiae. We have identified the common and different features between the three genomes in the protein evolution patterns. M. jannaschii has been seen to have a greater number of proteins with more charged amino acids whereas S. cerevisiae has been observed to have a greater number of hydrophilic proteins. Despite the differences in intrinsic compositional characteristics between the proteins from the different genomes we have also identified certain common characteristics. We have carried out exploratory Principal Component Analysis of the multivariate data on the proteins of each organism in an effort to classify the proteins into clusters. Interestingly, we found that most of the proteins in each organism cluster closely together, but there are a few 'outliers'. We focus on the outliers for the functional investigations, which may aid in revealing any unique features of the biology of the respective organisms

Archaeal Proteins↗
PubMed1999

Universal protein families and the functional content of the last universal common ancestor.

The phylogenetic distribution of Methanococcus jannaschii proteins can provide, for the first time, an estimate of the genome content of the last common ancestor of the three domains of life. Relying on annotation and comparison with reference to the species distribution of sequence similarities results in 324 proteins forming the universal family set. This set is very well characterized and relatively small and nonredundant, containing 301 biochemical functions, of which 246 are unique. This universal function set contains mostly genes coding for energy metabolism or information processing. It appears that the Last Universal Common Ancestor was an organism with metabolic networks and genetic machinery similar to those of extant unicellular organisms.

Archaeal Proteins↗
PubMed1999

Functional classes in the three domains of life.

The evolutionary divergence among the three major domains of life can now be addressed through the first set of complete genomes from representative species. These model species from the three domains of life, Haemophilus influenzae for Bacteria, Saccharomyces cerevisiae for Eukarya, and Methanococcus jannaschii for Archaea, provide the basis for a universal functional classification and analysis. We have chosen 13 functional classes and three superclasses (ENERGY, COMMUNICATION and INFORMATION) as global descriptors of protein function. Compositional comparison of the three complete genomes reveals that functional classes are ubiquitous yet diverse in the three domains of life. Proteins related with ENERGY processes are generally represented in all three domains, while those related with COMMUNICATION represent the most distinctive functional feature of each single domain. Finally, functions related with INFORMATION processing (translation, transcription, and replication) show a complex behaviour. In Archaea, proteins in this superclass are related with proteins in either Eukarya or Bacteria, as recognized previously. The distribution of functional classes in the three domains accurately reflects the principal characteristics of cellular life forms.

Archaeal Proteins↗
PubMed2000

A simplified method for the cultivation of extreme anaerobic Archaea based on the use of sodium sulfite as reducing agent.

The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development of archaeal genetic exchange systems. The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic Archaea but involves time-consuming procedures such as the use of air locks and glove boxes. We describe here a procedure for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without the use of an anaerobic chamber. When the reducing agent sodium sulfide (Na2S) was replaced by sodium sulfite (Na2SO3), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber. Exchange of the headspace of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media. Organisms that were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide. H2S was simply added to the headspace of serum bottles by a syringe. The use of H2S as a source for sulfur minimized the precipitation of cations by sulfide. Representatives of 12 genera of anaerobic Archaea studied here were able to grow in media prepared by this procedure. For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent. The application of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably.

Anaerobiosis↗
PubMed1997

ThiD-TenA: a gene pair fusion in eukaryotes.

Computational analysis of the hypothetical open reading frame MJ0236 from Methanococcus jannaschii reveals its membership to a family of bacterial and eukaryotic proteins, predicted to be the HMP-P kinases involved in thiamin biosyntheis (ThiD). The eukaryotic members of this family contain a C-terminal extension similar to a bacterial transcriptional activator (TenA), thus pointing to a fusion event that took place during cellular evolution. The C-terminal domain is absent from M. jannaschii. The significance of this observation is two-fold: first, this is a case where a fusion protein contains two domains with an unusual phylogenetic distribution, and second, the TenA domain is a rare case of a gene family involved in transcription present both in bacteria and eukaryotes.

Amino Acid Sequence↗