Biomedical subjects

A Böck

Publications and source records attributed to A Böck.

At least 55 records · Page 3Linked to original sources

PubMed1997

Selenoprotein synthesis in archaea: identification of an mRNA element of Methanococcus jannaschii probably directing selenocysteine insertion.

Selenocysteine is encoded by a UGA codon in all organisms that synthesise selenoproteins. This codon is specified as a selenocysteine codon by an mRNA secondary structure, which is located immediately 3' of the UGA in the reading frame of selenoprotein genes in Gram-negative bacteria, whereas it is located in the 3' untranslated region of eukaryal selenoprotein genes. The location and the structure of a similar mRNA signal in archaea has so far not been determined. Seven selenoproteins were identified for the archaeon Methanococcus jannaschii by labelling with 75Se and by SDS/polyacrylamide electrophoresis. Their size could be correlated with open reading frames possessing internal UGA codons from the total genomic sequence. One of the open reading frames, that of the VhuD subunit of a hydrogenase, possesses two UGA codons and appears to code for a selenoprotein with two selenocysteine residues. A strongly conserved mRNA element was identified that is exclusively linked to selenoprotein genes. It is located in the 3' untranslated region in six of the mRNAs and in the 5' untranslated region of the fdhA mRNA. This element, which is present in the 3' non-translated region of two selenoprotein mRNAs from Methanococcus voltae, is proposed to act in decoding of the UGA with selenocysteine.

Amino Acid Sequence↗

PubMed1997

The path of unspecific incorporation of selenium in Escherichia coli.

The path of unspecific selenium incorporation into proteins was studied in Escherichia coli mutants blocked in the biosynthesis of cysteine and methionine or altered in its regulation. Selenium incorporation required all enzymatic steps of cysteine biosynthesis except sulfite reduction, indicating that intracellular reduction of selenite occurs nonenzymatically. Cysteine (but not methionine) supplementation prevented unspecific incorporation of selenium by repressing cysteine biosynthesis. On the other hand, when the biosynthesis of cysteine was derepressed in regulatory mutants, selenium was incorporated to high levels. These findings and the fact that methionine auxotrophic strains still displayed unspecific incorporation show that selenium incorporation into proteins in E. coli occurs mainly as selenocysteine. These findings also provide information on the labeling conditions for incorporating 75Se only and specifically into selenoproteins.

Acetyltransferases↗

PubMed1997

Characterization of fhlA mutations resulting in ligand-independent transcriptional activation and ATP hydrolysis.

The FhlA protein belongs to the NtrC family of transcriptional regulators. It induces transcription from the -12/-24 promoters of the genes of the formate regulon by sigma54 RNA polymerase. FhlA is activated by binding of the ligand formate and does not require phosphorylation. A mutational analysis of the fhLA gene portion coding for the A and C domains was conducted with the aim of gaining information on the interaction between formate binding and ATP hydrolysis plus transcription activation. Four mutations were identified, all located in the A domain; one of them rendered transcription completely independent from the presence of formate, and the others conferred a semiconstitutive phenotype. The FhlA protein of one of the semiconstitutive variants was purified. Catalytic efficiency of ATP hydrolysis of the mutant FhlA was increased in the absence of formate in the same manner as formate influences the activity of wild-type FhlA. Moreover, in vitro transcription occurred at much lower threshold concentrations of the mutant protein and of nucleoside triphosphates than with the wild-type FhlA.

Adenosine Triphosphate↗

PubMed1997

Barriers to heterologous expression of a selenoprotein gene in bacteria.

The specificity parameters counteracting the heterologous expression in Escherichia coli of the Desulfomicrobium baculatum gene (hydV) coding for the large subunit of the periplasmic hydrogenase which is a selenoprotein have been studied. hydV'-'lacZ fusions were constructed, and it was shown that they do not direct the incorporation of selenocysteine in E. coli. Rather, the UGA codon is efficiently suppressed by some other aminoacyl-tRNA in an E. coli strain possessing a ribosomal ambiguity mutation. The suppression is decreased by the strA1 allele, indicating that the hydV selenocysteine UGA codon has the properties of a "normal" and suppressible nonsense codon. The SelB protein from D. baculatum was purified; in gel shift experiments, D. baculatum SelB displayed a lower affinity for the E. coli fdhF selenoprotein mRNA than E. coli SelB did and vice versa. Coexpression of the hydV'-'lacZ fusion and of the selB and tRNA(Sec) genes from D. baculatum, however, did not lead to selenocysteine insertion into the protein, although the formation of the quaternary complex between SelB, selenocysteyl-tRNA(Sec), and the hydV mRNA recognition sequence took place. The results demonstrate (i) that the selenocysteine-specific UGA codon is readily suppressed under conditions where the homologous SelB protein is absent and (ii) that apart from the specificity of the SelB-mRNA interaction, a structural compatibility of the quaternary complex with the ribosome is required.

Amino Acid Sequence↗

PubMed1997

Domain structure of the selenocysteine-specific translation factor SelB in prokaryotes.

Translation factor SelB is the key component for the specific decoding of UGA codons with selenocysteine at the ribosome. SelB binds selenocysteyl-tRNA(Sec), guanine nucleotides and a secondary structure of the selenoprotein mRNA following the UGA at the 3' side. A comparison of the amino acid sequences of SelB species from E. coli, Desulfomicrobium baculatum, Clostridium thermoaceticum and Haemophilus influenzae showed that the proteins consist of at least four structural domains from which the N-terminal three are well conserved and share homology with elongation factor Tu whereas the C-terminal one is more variable and displays no similarity to any protein known. With the aid of the coordinates of EF-Tu the N-terminal part has been modelled into a 3D structure which exhibits intriguing features concerning its interaction with guanine nucleotides and other components of the translational apparatus. Cloning and expression of fragments of SelB and biochemical analysis of the purified truncated proteins showed that the C-terminal 19 kDa protein fragment is able to specifically bind to the selenoprotein mRNA. SelB, thus, is a translation factor functionally homologous to EF-Tu hooked up to the mRNA with its C-terminal end. The formation by SelB of a quaternary complex in vivo has been proven by overexpression of truncated genes of SelB and by demonstration that fragments comprising the mRNA or the tRNA binding domain inhibit selenocysteine insertion.

Bacterial Proteins↗

PubMed1996

Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome.

The codon UGA located 5' adjacent to an mRNA hairpin within fdhF mRNA promotes the incorporation of the amino acid selenocysteine into formate dehydrogenase H of Escherichia coli. The loop region of this mRNA hairpin has been shown to bind to the special elongation factor SELB, which also forms a complex with selenocysteinyl-tRNA(Sec) and GTP. We designed seven different mRNA constructs derived from the fdhF mRNA which contain a translation initiation region including an AUG initiation codon followed by no, one, two, three, four, five or six UUC phenylalanine codon(s) and the UGA selenocysteine codon 5' adjacent to the fdhF mRNA hairpin. By binding these different mRNA constructs to 30S ribosomal subunits in vitro we attempted to mimic intermediate steps of elongation of a structured mRNA approaching the ribosome by one codon at a time. Toeprint analysis of the mRNA-ribosome complexes showed that the presence of the fdhF mRNA hairpin strongly interferes with binding of the fdhF mRNA to 30S ribosomal subunits as soon as the hairpin is placed closer than 16 bases to the ribosomal P-site. Binding is reduced up to 25-fold compared with mRNA constructs where the hairpin is located outside the ribosomal mRNA track. Surprisingly, no toeprint signals were observed in any of our mRNA constructs when tRNA(Sec) was used instead of tRNA(fMet). Lack of binding of selenocysteinyl-tRNA(Sec) to the UGA codon was attributed to steric hindrance by the fdhF mRNA hairpin. By chemical probing of the shortest mRNA construct (AUG-UGA-fdhF hairpin) bound to 30S ribosomal subunits we demonstrate that the hairpin structure is not unfolded in the presence of ribosomes in vitro; also, this mRNA is not translated in vivo when fused in-frame 5' of the lacZ gene. Therefore, our data indicate that the fdhF mRNA hairpin has to be unfolded during elongation prior to entering the ribosomal mRNA track and we propose that the SELB binding domain within the fdhF mRNA is located outside the ribosomal mRNA track during decoding of the UGA selenocysteine codon by the SELB-selenocysteinyl-tRNA(Sec)-GTP complex.

Aldehydes↗

PubMed1996

Domain structure of the prokaryotic selenocysteine-specific elongation factor SelB.

Incorporation of the non-canonical amino acid selenocysteine into proteins requires the activity of the elongation factor SelB which substitutes for the function of EF-Tu in contrast to EF-Tu, SelB binds selenocystylated tRNASec and an mRNA secondary structure adjacent to the UGA selenocysteine codon. To gain information on the domain structure of this specialized translation factor, the selB genes from two bacteria unrelated to Escherichia coli (Clostridium thermoaceticum and Desulfomicrobium baculatum) were cloned and sequenced. The derived amino acid residue sequences were compared to those of SelB from E. coli and Haemophilus influenzae and to EF-Tu sequences. The alignment revealed that SelB contains all three domains characterized for EF-Tu. A fourth, C-terminally located domain shows only limited sequence conservation within the four SelB proteins. To elucidate the function of this C-terminal part a structure-function analysis of SelB from E. coli was performed. It showed that a C-terminal 17 kDa subdomain of the translation factor, when expressed separately, specifically binds the mRNA secondary structure. The recognition motif itself could be reduced to a 17 nucleotide minihelix without loss of binding affinity and specificity. A truncated SelB lacking the mRNA binding domain was still able to interact with selenocysteyl-tRNASec. Expression of the mRNA binding domain alone suppressed selenocysteine insertion in vivo by competing with SelB for its binding site at the mRNA. The results indicate that SelB can be considered as an EF-Tu homolog hooked to the mRNA via its C-terminal domain.

Bacterial Proteins↗

PubMed1996

Generation of active [NiFe] hydrogenase in vitro from a nickel-free precursor form.

The maturation process of [NiFe] hydrogenases includes formation of the nickel metallocenter, proteolytic processing of the large subunit, and assembly with the other hydrogenase subunit(s). An in vitro system for the maturation of the large subunit (HycE) of hydrogenase 3 of Escherichia coli leading to an active enzyme was established. The system is based on extracts of an E. coli mutant lacking the nickel-specific transport system (nik). HycE was present in these extracts in the C-terminally extended precursor form devoid of nickel. Addition of nickel led to nickel incorporation and proteolytic processing of HycE. Under anaerobic conditions, hydrogenase 3 activity was subsequently generated. The maximal rate of the processing reaction was reached at a nickel concentration of 400 microM. The accessory proteins known to be involved in the maturation of HycE in vivo, namely HypB, HypC, HypD, HypE, HypF, and the protease HycI, are required for the in vitro reaction, since processing of HycE did not occur in extracts of double mutants affected in the nik system and in one of the accessory genes. Processing of HycE and generation of hydrogenase 3 activity were achieved in extracts of the nik- delta hycI mutant by addition of both nickel and purified HycI protease.

Bacterial Proteins↗

PubMed1996

On the mechanism of selenium tolerance in selenium-accumulating plants. Purification and characterization of a specific selenocysteine methyltransferase from cultured cells of Astragalus bisculatus.

Selected members of the genus Astragalus (Fabaceae) are known for their ability to accumulate high levels of selenium, mainly in the form of Se-methyl-selenocysteine. With the aid of cell cultures we have investigated the molecular basis for selenium tolerance of these plants. It is shown that cultured cells from a selenium-accumulating Astragalus species synthesize Se-methyl-selenocysteine in contrast to those of a non-accumulating species and do not unspecifically incorporate selenium into proteins. The purification and biochemical characterization of a selenocysteine methyltransferase from cultured Astragalus bisculatus cells is described, which does not accept cysteine as a substrate. We propose that this enzyme plays a crucial role in conferring selenium tolerance.

Adaptation, Physiological↗

PubMed1996

Genetics of a novel starch utilisation pathway present in Klebsiella oxytoca.

A 14.3 kb DNA fragment from Klebsiella oxytoca M5a1 has been cloned and shown to provide Escherichia coli with the capacity for growth on alpha- and beta-cyclodextrins. This fragment is located immediately upstream of the previously identified cgt gene coding for cyclodextrin glycosyltransferase. It contains ten genes (cym) organised in two divergently oriented clusters separated by a non-coding region of 419 bp. Four of the genes code for products homologous to the maltose and linear maltodextrin uptake system, another one for a putative cytoplasmic cyclodextrinase. The cym genes of K. oxytoca are distinct and different from the mal genes; cym mutations do not affect maltose catabolism. On the other hand, whereas mutations in the maltose/maltodextrin-uptake genes do not influence cyclodextrin metabolism, a mutation inactivating the malPQ genes coding for maltodextrin phosphorylase and amylomaltase does. Cyclodextrin catabolism is independent of the presence of a functional cyclodextrin glycosyltransferase but degradation of starch and gamma-cyclodextrins requires the activity of this enzyme. The results indicate the existence of a novel starch degradation pathway which involves the extracellular conversion of starch into cyclodextrins by cyclodextrin glycosyltransferase, uptake of the cyclodextrins by a specific uptake system and intracellular linearisation by a cyclodextrinase. The malto-oligosaccharides produced are then channelled into the maltodextrin-degradation route involving the activity of maltodextrin phosphorylase and amylomaltase.

Amino Acid Sequence↗

PubMed1996

Metabolism of cyclodextrins by Klebsiella oxytoca m5a1: purification and characterisation of a cytoplasmically located cyclodextrinase.

It has been shown previously that the products of 11 genes are required for metabolism of starch by Klebsiella oxytoca via a novel pathway. An extracellular cyclodextrin glucanotransferase first degrades starch into alpha- and beta-cyclodextrins; evidence then has been presented that the cyclodextrins are transported into the cytoplasma via a specific system and that they are metabolised inside the cell. To provide support for this model, we have analysed whether Klebsiella oxytoca possesses a cytoplasmic enzyme able to linearise cyclodextrins. A possible candidate was the product of the cymH gene since it displays sequence similarity with cyclodextrinases from other organisms. The cymH gene was overexpressed, and the CymH protein was purified. CymH is a monomer of 69 kDa molecular mass and hydrolysed cyclodextrins at an optimum pH of 7.0 and an optimum temperature of 23 degrees C, respectively. The apparent Km increased with increasing size of the cyclodextrins, but the reaction velocity decreased. Linear malto-oligosaccharides were also accepted as substrates, but were hydrolysed with a lower efficiency. Final products in each case were maltose and maltotriose. It was demonstrated by immunoblotting that CymH is located in the cytoplasm and that no signal peptide was cleaved off. Since cymH mutants were no longer able to grow on cyclodextrins, these results prove that cyclodextrins are degraded inside the cell, and they support the contention of the existence of a specific transport system.

Base Sequence↗

PubMed1996

Nickel incorporation into hydrogenase 3 from Escherichia coli requires the precursor form of the large subunit.

A mutant derivative of hycE, the gene for the large subunit of hydrogenase 3 from Escherichia coli, was constructed that lacks the 3'-terminal part encoding the C-terminal portion of the HycE polypeptide, which is proteolytically removed during maturation of the hydrogenase. The truncated gene was transferred to the in situ position on the chromosome. Although the mutant possessed HycE in its "mature" form, it was devoid of hydrogenase 3 activity. The activity was not restored by high nickel concentrations in the medium. The mutated HycE was not associated with detectable radioactivity when the strain was grown in the presence of 63Ni2+. These results indicate that the C-terminal extension in the precursor form of the large subunit keeps the protein in a conformation required for the coordination of the metal.

Base Sequence↗

PubMed1996

Analysis of the hydA locus of Escherichia coli: two genes (hydN and hypF) involved in formate and hydrogen metabolism.

The hydA locus of Escherichia coli is known to encode some function necessary for formation of hydrogenase activity. The locus contains two open reading frames, hydN and hypF. In this communication, an analysis of the regulation of these two genes and of the phenotype of respective mutants is presented, Both genes were expressed in a T7 promoter/polymerase system, yielding a 19-kDa (HydN) and a 81-kDa (HypF) protein. In-frame deletions were constructed for each gene and transferred to the chromosome by homologous recombination. The mutation in hydN led to a decrease of the activity of formate dehydrogenase H (FDH-H) in crude extracts, but the activity and maturation of hydrogenases were nearly unaffected. In contrast, a deletion in hypF resulted in the loss of hydrogenase activity and in the synthesis of the large subunits of the hydrogenase isoenzymes 1, 2 and 3 in the inactive precursor form. For hydrogenase 3, it was shown that this is due to a lack of incorporation of nickel into the large subunit. hydN and hypF are organised in an operon that is a member of the formate regulon. Transcription was shown to be dependent on sigma 54 and FhlA, and an FhlA-binding site upstream of hydN was identified. The sigma 54-dependent promoter shows a rare deviation from the consensus at positions -24/-12, namely GG/GA instead of GG/GC. In conclusion, the product of hydN appears to have some role in electron flow from or to FDH-H, and the product of hypF is connected with maturation of all three hydrogenases of E. coli.

Amino Acid Sequence↗

PubMed1996

Structural model for the selenocysteine-specific elongation factor SelB.

A structural model was established for the N-terminal part of translation factor SelB which shares sequence similarity with EF-Tu, taking into account the coordinates of the EF-Tu 3D structure and the consensus of SelB sequences from four bacteria. The model showed that SelB is homologous in its N-terminal domains over all three domains of EF-Tu. The guanine nucleotide binding site and the residues involved in GTP hydrolysis are similar to those of EF-Tu, but with some subtle differences possibly responsible for the higher affinity of SelB for GTP compared to GDP. In accordance, the EF-Tu epitopes interacting with EF-Ts are lacking in SelB. Information on the formation of the selenocysteyl-binding pocket is presented. A phylogenetic comparison of the SelB domains homologous to EF-Tu with those from EF-Tu and initiation factor 2 indicated that SelB forms a separate class of translation factors.

Amino Acid Sequence↗

PubMed1996

Role of stoichiometry between mRNA, translation factor SelB and selenocysteyl-tRNA in selenoprotein synthesis.

The specialized translation factor SelB forms a quaternary complex in vitro with selenocysteyl-tRNA(Sec), the selenoprotein mRNA and guanine nucleotides. To gain information on whether this complex is required for selenocysteine insertion in vivo we have studied the effect of unbalanced ratios of the individual components of the complex on UGA readthrough. It was found that overproduction of SelB in an otherwise wild-type genetic background reduced UGA readthrough to less than 1%. Concomitant overexpression of selC (the gene for selenocysteine-specific tRNA(Sec)) completely reversed the inhibition. Truncation of SelB from the C-terminal end abolished function as a translation factor but the truncated molecules, when overproduced, were still able to suppress UGA read-through. The inhibition was also reversed by overproduction of tRNA(Sec). The most plausible explanation is that overproduction of SelB impairs the statistics of formation of the quaternary complex and that the C-terminally truncated molecules are still able to bind selenocysteyl-tRNA(Sec) and remove it from the pool. The mRNA-binding capacity, therefore, is physically separated from the selenocysteyl-tRNA-binding domain.

Bacteria↗

PubMed1996

The nucleotide concentration determines the specificity of in vitro transcription activation by the sigma 54-dependent activator FhlA.

An in vitro transcription system has been set up for formate- and FhlA-dependent transcription activation at the -12/-24 promoter of the fdhF gene from Escherichia coli by sigma 54-RNA polymerase. It requires the presence of the upstream activation sequence on supercoiled DNA. Transcription is independent from the effector formate at nucleoside triphosphate concentrations of 400 microM and above and completely dependent on the presence of the effector when the concentration is lowered to 300 microM. Inclusion of nucleoside diphosphates in the system raises the nucleoside triphosphate level at which specific induction by formate can take place. The threshold level of FhlA relative to that of template DNA required for transcription activation in the absence of formate was lowered at a high nucleoside triphosphate concentration. On the other hand, transcription activation at the fdhF promoter lacking the upstream activation sequence requires an increased ratio of FhlA to promoter plus the presence of formate; high ATP concentrations cannot bypass the effect of formate. These results are interpreted in terms of a model which implies that FhlA must undergo a change in its oligomeric state for transcription activation and that this oligomerization is favored by high nucleoside triphosphate concentrations, by the effector formate, and by the target DNA. In the absence of the target DNA, FhlA can line up at unspecific DNA and activate transcription; in this case, however, presence of formate and a higher FhlA concentration are required to stabilize and increase the amount of active oligomer.

Base Sequence↗

PubMed1996

Solution structure of mRNA hairpins promoting selenocysteine incorporation in Escherichia coli and their base-specific interaction with special elongation factor SELB.

On the basis of chemical probing data, the solution structures of RNA hairpins within fdhF and fdnG mRNAs in Escherichia coli, which both promote selenocysteine incorporation at UGA codons, were derived with the help of computer modeling. We find that these mRNA hairpins contain two separate structural domains that possibly also exert two different functions. The first domain is comprised of the UGA codon, which is included within a complex and distorted double-stranded region. Thereby, release factor 2 might be prevented from binding to the UGA codon to terminate protein synthesis. The second domain is located within the apical loop of the mRNA hairpin structures. This loop region exhibits a defined tertiary structure in which no base is involved in Watson-Crick interactions. The structure of the loop is such that, following a sharp turn after G22 (A22 in fdnG mRNA), bases G23 and U24 are exposed to the solvent on the deep groove side of the supporting helix. Residues C25 and U26 close the loop with a possible single H-bonding interaction between the first and last residues of the loop, 04(U26) and N6(A21). The bulge residues U17 and U18 (in fdhF mRNA), or Ul7 only in fdnG mRNA, point their Watson-Crick positions in the same direction as loop residues G23 and U24 do, and at the same time open up the deep groove at the top of the hairpin helix. Chemical probing data demonstrate that bases G23 and U24 in both mRNA hairpins, as well as residues U17 and Ul7/U18 (for fdhF mRNA) located in a bulge 5' to the loop, are involved directly in binding to special elongation factor SELB in both mRNAs. Therefore, SELB recognizes identical bases within both mRNA hairpins despite differences in their primary sequence, consistent with the derived 3D models for these mRNAs, which exhibit similar tertiary structures. Binding of SELB to the fdhF mRNA hairpin was estimated to proceed with an apparent Kd of 30 nM.

Aldehydes↗

PubMed1995

GTP hydrolysis by HypB is essential for nickel insertion into hydrogenases of Escherichia coli.

The product of the hypB gene, which is required for the maturation of the three [NiFe]hydrogenases of Escherichia coli, is a member of the GTPase family and exhibits a low intrinsic GTPase activity. It was studied whether or not GTP hydrolysis by HypB is coupled to nickel insertion into hydrogenases and to maturation of hydrogenases. Mutations were introduced into the hypB gene at sites expected to code for amino acids involved in guanine-nucleotide binding. Lys117 of G-motif 1, as well as Asp241 of G-motif 4 were substituted by asparagine residues. The purified mutant HypB proteins showed strongly reduced, but still significant, GTPase activity. In the case of [D241N]HypB, the kcat/Km value was lowered by a factor of 85 and the specificity of the enzyme for GTP was apparently lost, with other nucleoside triphosphates including XTP becoming compatible substrates. The decrease in GTPase activity was even more pronounced for [K117N]HypB. To assess the functionality of these HypB proteins in vivo, the wild-type hypB gene in the chromosome of E. coli was replaced by the mutant alleles. The resulting mutant strains BKN117 and BDN241 were affected in hydrogen metabolism under fermentative conditions. BKN117 did not display hydrogenase activity due to a loss of nickel incorporation into the large subunit. BDN241 exhibited a reduction of hydrogenase activity by 44% and only a portion of the hydrogenase 3 large subunit was in the mature nickel-containing form. From these results, it is concluded that GTP hydrolysis catalysed by HypB is an integral process in nickel incorporation into hydrogenases.

Amino Acid Sequence↗