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PubMed2003

Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression.

Recently, it has been shown that an amber suppressor tRNA/aminoacyl-tRNA synthetase pair derived from the tyrosyl-tRNA synthetase of Methanococcus jannaschii can be used to genetically encode unnatural amino acids in response to the amber nonsense codon, TAG. However, we have been unable to modify this pair to decode either the opal nonsense codon, TGA, or the four-base codon, AGGA, limiting us to a 21 amino acid code. To overcome this limitation, we have adapted a leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum and leucyl tRNA derived from Halobacterium sp. NRC-1 as an orthogonal tRNA-synthetase pair in Escherichia coli to decode amber (TAG), opal (TGA), and four-base (AGGA) codons. To improve the efficiency and selectivity of the suppressor tRNA, extensive mutagenesis was performed on the anticodon loop and acceptor stem. The two most significant criteria required for an efficient amber orthogonal suppressor tRNA are a CU(X)XXXAA anticodon loop and the lack of noncanonical or mismatched base pairs in the stem regions. These changes afford only weak suppression of TGA and AGGA. However, this information together with an analysis of sequence similarity of multiple native archaeal tRNA sequences led to efficient, orthogonal suppressors of opal codons and the four-base codon, AGGA. Ultimately, it should be possible to use these additional orthogonal pairs to genetically incorporate multiple unnatural amino acids into proteins.

Anticodon↗
PubMed2005

Electrostatic interaction of a K+ channel RCK domain with charged membrane surfaces.

In a subset of K(+) channels, gating is regulated through the direct binding of ligands by large cytoplasmic RCK domains. To further investigate the role of the RCK domain, we have begun the biochemical characterization of a two-transmembrane segment, RCK domain-containing channel from Methanococcus jannaschii, MjK2, by testing its general functional behavior and identifying purification conditions. Standard detergent solubilization of recombinantly expressed MjK2 required the addition of a high NaCl concentration to the extraction buffer for MjK2 solubilization. The cytoplasmic RCK domain was identified as the region of MjK2 responsible for the dependence of solubilization on high salt concentrations since expression of an MjK2 construct lacking the transmembrane domain, MjK2cd, also required high salt concentrations for extraction from Escherichia coli lipids, a necessary step in the purification of both MjK2 and MjK2cd. MjK2 expression was able to weakly recover growth of K(+) uptake deficient LB2003 cells at 10 mM KCl, suggesting that the channel can conduct K(+) but has a low open probability. Purified MjK2 reconstituted in liposomes generated only limited Rb(+) uptake, blocked by BaCl(2). MjK2cd exhibited direct binding to PC/PS lipid vesicles with a molar partition coefficient (K(1)) of approximately 10(3) M(-)(1), which decreased with both an increase in the salt concentration and a decrease in the anionic lipid ratio. Lipid blot assays revealed that MjK2cd binds most strongly to lipids of increasingly negative charge. These results support the idea that the binding of the MjK2 RCK domain to membranes takes place via an electrostatic interaction with anionic lipid surfaces.

Amino Acid Sequence↗
PubMed2005

Structural and functional investigation of a putative archaeal selenocysteine synthase.

Bacterial selenocysteine synthase converts seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec) for selenoprotein biosynthesis. The identity of this enzyme in archaea and eukaryotes is unknown. On the basis of sequence similarity, a conserved open reading frame has been annotated as a selenocysteine synthase gene in archaeal genomes. We have determined the crystal structure of the corresponding protein from Methanococcus jannaschii, MJ0158. The protein was found to be dimeric with a distinctive domain arrangement and an exposed active site, built from residues of the large domain of one protomer alone. The shape of the dimer is reminiscent of a substructure of the decameric Escherichia coli selenocysteine synthase seen in electron microscopic projections. However, biochemical analyses demonstrated that MJ0158 lacked affinity for E. coli seryl-tRNA(Sec) or M. jannaschii seryl-tRNA(Sec), and neither substrate was directly converted to selenocysteinyl-tRNA(Sec) by MJ0158 when supplied with selenophosphate. We then tested a hypothetical M. jannaschii O-phosphoseryl-tRNA(Sec) kinase and demonstrated that the enzyme converts seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec) that could constitute an activated intermediate for selenocysteinyl-tRNA(Sec) production. MJ0158 also failed to convert O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). In contrast, both archaeal and bacterial seryl-tRNA synthetases were able to charge both archaeal and bacterial tRNA(Sec) with serine, and E. coli selenocysteine synthase converted both types of seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). These findings demonstrate that a number of factors from the selenoprotein biosynthesis machineries are cross-reactive between the bacterial and the archaeal systems but that MJ0158 either does not encode a selenocysteine synthase or requires additional factors for activity.

Amino Acid Sequence↗
PubMed2006

The temperature dependence of the inositol monophosphatase Km correlates with accumulation of di-myo-inositol 1,1'-phosphate in Archaeoglobus fulgidus.

Di-myo-inositol 1,1'-phosphate (DIP) accumulates as a compatible solute in many hyperthermophilic archaea (e.g., Archaeoglobus fulgidus) when the cells are grown above 80 degrees C. Recent microarray analysis of A. fulgidus transcripts [Rohlin, L., et al. (2005) J. Bacteriol. 187, 6046] indicates that neither the myo-inositol-1-phosphate synthase, the first step in inositol biosynthesis, nor the inositol monophosphatase (IMPase), which generates myo-inositol, are significantly upregulated upon thermal stress. Although other factors could contribute to regulation of DIP synthesis in cells, there is an 8-10-fold decrease in the K(m) of the IMPase for inositol phosphates between 75 and 85 degrees C (for l-I-1-P, the K(m) decreased from 13.2 to 1.67 mM) that correlates with the observed accumulation of DIP in cells. Between 55 and 75 degrees C, K(m) values decreased 2.3-fold at most. The enzyme also exhibits fructose bisphosphatase activity. However, the K(m) for fructose 1,6-bisphosphate was low and the same (0.15 +/- 0.01 mM) at 55 and 70 degrees C. This indicates that the unusual temperature dependence of K(m) is specific for I-1-P substrates. (31)P NMR studies confirmed that the affinity of inositol 1-phosphate for the enzyme was indeed weak (K(D) >or= 5 mM) below but increased significantly at 80 degrees C. In contrast, the IMPase from Methanococcus jannaschii, an organism in which DIP does not accumulate, had a low K(m) for I-1-P over the entire temperature range. A structural comparison of the two archaeal IMPases identified a hydrogen bonding network present in the active site of the A. fulgidus enzyme and not in the M. jannaschii IMPase, the disruption (e.g., A. fulgidus IMPase S171A or T174L) of which prevented the drop in K(m) at high temperatures. We suggest that the temperature-dependent synthesis and accumulation of DIP in A. fulgidus are regulated in part by the temperature dependence of the K(m) of the IMPase activity in the cells.

Amino Acids↗
PubMed1999

N1-(5'-phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase: purification and characterization of a unique metalloenzyme.

N1-(5'-Phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase (HisI, PR-AMP cyclohydrolase) is a central enzyme in histidine biosynthesis catalyzing the hydrolysis of the N1-C6 bond of the purine substrate, a reaction unique to this pathway. A source of the recombinant monofunctional Methanococcus vannielii PR-AMP cyclohydrolase has been developed, and the first characterization of a purified form of the enzyme is reported. The enzyme has a native molecular weight of 31 200 as determined by analytical ultracentrifugation that agrees with the molecular mass determined by gel filtration (34 kDa) and a subunit molecular weight of 15 486 based on MALDI-MS. An unusual characteristic of the protein is the complexity observed on SDS-PAGE, and N-terminal amino acid sequence analysis of all the isolated constituents confirms their origin as PR-AMP cyclohydrolase. A highly conserved region of the amino acid sequence is implicated in the self-cleavage events of the protein and provides an explanation for the complexity of this protein. Bound to the enzyme is 1 equiv of Zn2+ that can be removed only by extended dialysis with 1,10-phenanthroline (Kd </= 10(-)9 M). Removal of the Zn2+ correlates with the loss of enzyme activity. The enzyme is reversibly inhibited by inclusion of EDTA in the assay mixture, demonstrating that free Mg2+ (Ks = 4.9 +/- 0.7 microM) is required for catalytic activity. Further evidence for a low-affinity binding site is indicated by the inhibitory effects of exogenous Zn2+ on enzyme activity. The pH dependence of the PR-AMP cyclohydrolase activity shows a single titration event in the kcat/Km profile with a pKa of 7.3 that is consistent with the functional role of a metal site in catalysis. These data are discussed in the context of the mechanism of other nucleotide hydrolases.

Amino Acid Sequence↗
PubMed2000

Identity of tRNA for yeast tyrosyl-tRNA synthetase: tyrosylation is more sensitive to identity nucleotides than to structural features.

The specific aminoacylation of tRNA by yeast tyrosyl-tRNA synthetase does not rely on the presence of modified residues in tRNA(Tyr), although such residues stabilize its structure. Thus, the major tyrosine identity determinants were searched by the in vitro approach using unmodified transcripts produced by T7 RNA polymerase. On the basis of the tyrosylation efficiency of tRNA variants, the strongest determinants are base pair C1-G72 and discriminator residue A73 (the 5'-phosphoryl group on C1, however, is unimportant for tyrosylation). The three anticodon bases G34, U35, and A36 contribute also to the tyrosine identity, but to a lesser extent, with G34 having the most pronounced effect. Mutation of the GUA tyrosine anticodon into a CAU methionine anticodon, however, leads to a loss of tyrosylation efficiency similar to that obtained after mutation of the C1-G72 or A73 determinants. Transplantation of the six determinants into four different tRNA frameworks and activity assays on heterologous Escherichia coli and Methanococcus jannaschii tRNA(Tyr) confirmed the completeness of the tyrosine set and the eukaryotic character of the C1-G72 base pair. On the other hand, it was found that tyrosine identity in yeast does not rely on fine architectural features of the tRNA, in particular the size and sequence of the D-loop. Noticeable, yeast TyrRS efficiently charges a variant of E. coli tRNA(Tyr) with a large extra-region provided its G1-C72 base pair is changed to a C1-G72 base pair. Finally, tyrosylation activity is compatible with a +1 shift of the anticodon in the 3'-direction but is strongly inhibited if this shift occurs in the opposite 5'-direction.

Acylation↗
PubMed2001

Isolation, crystallization, and investigation of ribosomal protein S8 complexed with specific fragments of rRNA of bacterial or archaeal origin.

The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8-RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.

Binding Sites↗
PubMed2001

The nodulin vfENOD18 is an ATP-binding protein in infected cells of Vicia faba L. nodules.

Recently we described the novel nodulin gene VfENOD18, whose corresponding transcripts were restricted to the nitrogen-fixing zone III of broad bean root nodules. To characterize VfENOD18 on the protein level, polyclonal antibodies were generated using the purified recombinant VfENOD18 protein produced in Escherichia coli by employing the pMAL-c expression system. These antibodies recognized immunoreactive proteins isolated from indeterminate nodules of different leguminous plants, but also from non-symbiotic tissues of Glycine max and from tissues of Arabidopsis thaliana and Zea mays. Using immunogold labelling the nodulin VfENOD18 was localized to the cytoplasm of infected cells in the nitrogen-fixing zone of broad bean nodules. Due to the homology of the VfENOD18 sequence to that of the ATP-binding protein MJ0577 from the hyperthermophile Methanococcus jannaschii the recombinant VfENOD18 protein was tested for ATP-binding. Using the biotin photoaffinity ATP analogue 8N3ATP[gamma]biotin it could be demonstrated that VfENOD18 is an ATP-binding protein. PCR experiments revealed that the amino acid sequences of the putative C-terminal ATP-binding sites of the VfENOD 18 homologues from Lens culinaris, Vicia hirsuta, Vicia sativa and Vicia villosa were conserved. We propose that VfENOD18 is a member of a novel family of ATP-binding proteins in plants.

Adenosine Triphosphate↗
PubMed1998

Crystal structure of a small heat-shock protein.

The principal heat-shock proteins that have chaperone activity (that is, they protect newly made proteins from misfolding) belong to five conserved classes: HSP100, HSP90, HSP70, HSP60 and the small heat-shock proteins (sHSPs). The sHSPs can form large multimeric structures and have a wide range of cellular functions, including endowing cells with thermotolerance in vivo and being able to act as molecular chaperones in vitro; sHSPs do this by forming stable complexes with folding intermediates of their protein substrates. However, there is little information available about these structures or the mechanism by which substrates are protected from thermal denaturation by sHSPs. Here we report the crystal structure of a small heat-shock protein from Methanococcus jannaschii, a hyperthermophilic archaeon. The monomeric folding unit is a composite beta-sandwich in which one of the beta-strands comes from a neighbouring molecule. Twenty-four monomers form a hollow spherical complex of octahedral symmetry, with eight trigonal and six square 'windows'. The sphere has an outer diameter of 120 A and an inner diameter of 65 A.

Amino Acid Sequence↗
PubMed1987

A possible biochemical missing link among archaebacteria.

Until recently all archaebacteria isolated conformed to one of three basic phenotypes: they were either methanogens, extreme halophiles, or ('sulphur-dependent') extreme thermophiles. However, a novel phenotype, that fits none of these categories, has recently been described. The organism, strain VC-16 (tentatively called "Archaeoglobus fulgidus") reduces sulphate--the only archaebacterium so far known to do so--and makes very small quantities of methane, although it lacks some of the cofactors normally associated with methanogenesis. These characteristics suggest that strain VC-16 might represent a transition form between an anaerobic thermophilic sulfur-based type of metabolism (which seems to be the ancestral metabolism for archaebacteria and methanogenesis (which somehow then derives from it). We here show that the lineage represented by strain VC-16 arises from the archaebacterial tree precisely where such an interpretation would predict that it would, between the Methanococcus lineage (which is the deepest of the methanogen branchings) and that of Thermococcus (the deepest of all branchings on the methanogen side of the tree).

Archaea↗
PubMed1998

Crystal structure of the bacterial cell-division protein FtsZ.

Bacterial cell division ends with septation, the constriction of the cell wall and cell membranes that leads to the formation of two daughter cells. During septation, FtsZ, a protein of relative molecular mass 40,000 which is ubiquitous in eubacteria and is also found in archaea and chloroplasts, localizes early at the division site to form a ring-shaped septum. This septum is required for the mechanochemical process of membrane constriction. FtsZ is a GTPase with weak sequence homology to tubulins. The nature of FtsZ polymers in vivo is unknown, but FtsZ can form tubules, sheets and minirings in vitro. Here we report the crystal structure at 2.8 A resolution of recombinant FtsZ from the hyperthermophilic methanogen Methanococcus jannaschii. FtsZ has two domains, one of which is a GTPase domain with a fold related to one found in the proteins p21ras and elongation factor EF-Tu. The carboxy-terminal domain, whose function is unknown, is a four-stranded beta-sheet tilted by 90 degrees against the beta-sheet of the GTPase domain. The two domains are arranged around a central helix. GDP binding is different from that typically found in GTPases and involves four phosphate-binding loops and a sugar-binding loop in the first domain, with guanine being recognized by residues in the central connecting helix. The three-dimensional structure of FtsZ is similar to the structure of alpha- and beta-tubulin.

Amino Acid Sequence↗
PubMed2000

PAN, the proteasome-activating nucleotidase from archaebacteria, is a protein-unfolding molecular chaperone.

The proteasome-activating nucleotidase (PAN) from Methanococcus jannaschii is a complex of relative molecular mass 650,000 that is homologous to the ATPases in the eukaryotic 26S proteasome. When mixed with 20S archaeal proteasomes and ATP, PAN stimulates protein degradation. Here we show that PAN reduces aggregation of denatured proteins and enhances their refolding. These processes do not require ATP hydrolysis, although ATP binding enhances the ability of PAN to prevent aggregation. PAN also catalyses the unfolding of the green fluorescent protein with an 11-residue ssrA extension at its carboxy terminus (GFP11). This unfolding requires ATP hydrolysis, and is linked to GFP11 degradation when 20S proteasomes are also present. This unfolding activity seems to be essential for ATP-dependent proteolysis, although PAN may function by itself as a molecular chaperone.

Adenosine Diphosphate↗
PubMed2001

An aminoacyl tRNA synthetase whose sequence fits into neither of the two known classes.

Aminoacyl transfer RNA synthetases catalyse the first step of protein synthesis and establish the rules of the genetic code through the aminoacylation of tRNAs. There is a distinct synthetase for each of the 20 amino acids and throughout evolution these enzymes have been divided into two classes of ten enzymes each. These classes are defined by the distinct architectures of their active sites, which are associated with specific and universal sequence motifs. Because the synthesis of aminoacyl-tRNAs containing each of the twenty amino acids is a universally conserved, essential reaction, the absence of a recognizable gene for cysteinyl tRNA synthetase in the genomes of Archae such as Methanococcus jannaschii and Methanobacterium thermoautotrophicum has been difficult to interpret. Here we describe a different cysteinyl-tRNA synthetase from M. jannaschii and Deinococcus radiodurans and its characterization in vitro and in vivo. This protein lacks the characteristic sequence motifs seen in the more than 700 known members of the two canonical classes of tRNA synthetase and may be of ancient origin. The existence of this protein contrasts with proposals that aminoacylation with cysteine in M. jannaschii is an auxiliary function of a canonical prolyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1999

Protein interaction maps for complete genomes based on gene fusion events.

A large-scale effort to measure, detect and analyse protein-protein interactions using experimental methods is under way. These include biochemistry such as co-immunoprecipitation or crosslinking, molecular biology such as the two-hybrid system or phage display, and genetics such as unlinked noncomplementing mutant detection. Using the two-hybrid system, an international effort to analyse the complete yeast genome is in progress. Evidently, all these approaches are tedious, labour intensive and inaccurate. From a computational perspective, the question is how can we predict that two proteins interact from structure or sequence alone. Here we present a method that identifies gene-fusion events in complete genomes, solely based on sequence comparison. Because there must be selective pressure for certain genes to be fused over the course of evolution, we are able to predict functional associations of proteins. We show that 215 genes or proteins in the complete genomes of Escherichia coli, Haemophilus influenzae and Methanococcus jannaschii are involved in 64 unique fusion events. The approach is general, and can be applied even to genes of unknown function.

Artificial Gene Fusion↗
PubMed2000

Crystal structures of ribosome anti-association factor IF6.

Ribosome anti-association factor eIF6 (originally named according to translation initiation terminology as eukaryotic initiation factor 6) binds to the large ribosomal subunit, thereby preventing inappropriate interactions with the small subunit during initiation of protein synthesis. We have determined the X-ray structures of two IF6 homologs, Methanococcus jannaschii archaeal aIF6 and Sacchromyces cerevisiae eIF6, revealing a phylogenetically conserved 25 kDa protein consisting of five quasi identical alpha/beta subdomains arrayed about a five-fold axis of pseudosymmetry. Yeast eIF6 prevents ribosomal subunit association. Comparative protein structure modeling with other known archaeal and eukaryotic homologs demonstrated the presence of two conserved surface regions, one or both of which may bind the large ribosomal subunit.

Amino Acid Sequence↗
PubMed2002

Structure of the SRP19 RNA complex and implications for signal recognition particle assembly.

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein. It associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. In mammalian cells, the SRP consists of a 7S RNA and six protein components. The S domain of SRP comprises the 7S.S part of RNA bound to SRP19, SRP54 and the SRP68/72 heterodimer; SRP54 has the main role in recognizing signal sequences of nascent polypeptide chains and docking SRP to its receptor. During assembly of the SRP, binding of SRP19 precedes and promotes the association of SRP54 (refs 4, 5). Here we report the crystal structure at 2.3 A resolution of the complex formed between 7S.S RNA and SRP19 in the archaeon Methanococcus jannaschii. SRP19 bridges the tips of helices 6 and 8 of 7S.S RNA by forming an extensive network of direct protein RNA interactions. Helices 6 and 8 pack side by side; tertiary RNA interactions, which also involve the strictly conserved tetraloop bases, stabilize helix 8 in a conformation competent for SRP54 binding. The structure explains the role of SRP19 and provides a molecular framework for SRP54 binding and SRP assembly in Eukarya and Archaea.

Amino Acid Sequence↗
PubMed2003

X-ray structure of a protein-conducting channel.

A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2 A. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The alpha-subunit has two linked halves, transmembrane segments 1-5 and 6-10, clamped together by the gamma-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel into an 'hourglass' with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocating polypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequence recognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates binding sites for partners that provide the driving force for translocation.

Archaeal Proteins↗
PubMed2003

An archaeal endoribonuclease catalyzes cis- and trans- nonspliceosomal splicing in mouse cells.

The tRNA endonuclease from the archaebacterium Methanococcus jannaschii (MJ endonuclease) can cleave RNAs forming specific bulge-helix-bulge (BHB) structures recognized by the enzyme. The resulting cleavage products are subsequently joined together by an endogenous ligase. We demonstrate the potential of using this strategy for repairing RNA in higher organisms by expressing the enzyme in mouse cells. Reporter target mRNAs modified with 17-nucleotide introns, flanked by sequences capable of forming BHB structures in cis, were expressed in mouse cells. RNA molecules that can form BHB substrates in trans with targeted mRNAs were also designed. Co-transfection of mouse cells with plasmids expressing these RNAs and the MJ endonuclease led to formation of RNA chimeras in which the target and exogenous RNA were recombined across the BHB. This technology is not limited to mRNA, but could in principle be used to destroy, modify or restore the function of a vast repertoire of RNA species or to join selectable tags to target RNAs.

3T3 Cells↗