Biomedical subjects

R Wilting

Publications and source records attributed to R Wilting.

10 recordsLinked to original sources

PubMed2006

Colanic acid is an exopolysaccharide common to many enterobacteria isolated from paper-machine slimes.

In this study, polysaccharide-producing bacteria were isolated from slimes collected from two Finnish and one Spanish paper mill and the exopolysaccharides (EPSs) produced by 18 isolates were characterised. Most of the isolates, selected on the bases of slimy colony morphology, were members of the family Enterobacteriaceae most frequently belonging to the genera Enterobacter and Klebsiella including Raoultella. All of the EPSs analysed showed the presence of charged groups in the form of uronic acid or pyruvate revealing the polyanionic nature of these polysaccharides. Further results of the carbohydrate analysis showed that the EPS produced by nine of the enterobacteria was colanic acid.

Biofilms↗

PubMed2001

Selenoprotein synthesis in archaea.

The availability of the genome sequences from several archaea has facilitated the identification of the encoded selenoproteins and also of most of the components of the machinery for selenocysteine biosynthesis and insertion. Until now, selenoproteins have been identified solely in species of the genera Methanococcus (M.) and Methanopyrus. Apart from selenophosphate synthetase, they include only enzymes with a function in energy metabolism. Like in bacteria and eukarya, selenocysteine insertion is directed by a UGA codon in the mRNA and involves the action of a specific tRNA and of selenophosphate as the selenium donor. Major differences to the bacterial system, however, are that no homolog for the bacterial selenocysteine synthase was found and, especially, that the SECIS element of the mRNA is positioned in the 3' nontranslated region. The characterisation of a homolog for the bacterial SelB protein showed that it does not bind to the SECIS element necessitating the activity of at least a second protein. The use of the genetic system of M. maripaludis allowed the heterologous expression of a selenoprotein gene from M. jannaschii and will facilitate the elucidation of the mechanism of the selenocysteine insertion process in the future.

Amino Acid Sequence↗

PubMed2000

Identification and characterisation of the selenocysteine-specific translation factor SelB from the archaeon Methanococcus jannaschii.

Selenocysteine insertion into archaeal selenopolypeptides is directed through an mRNA structure (the SECIS element) situated in the 3' non-translated region like in eukaryotes. To elucidate the mechanism how this element affects decoding of an in-frame UGA with selenocysteine the open reading frames of the genome of Methanococcus jannaschii were searched for the existence of a homolog to the bacterial specialized translation factor SelB. The product of the open reading frame MJ0495 was identified as the archaeal SelB homolog on the basis of the following characteristics: (1) MJ0495 possesses sequence features characteristic of bacterial SelB; (2) purified MJ0495 displays guanine nucleotide binding properties like SelB; and (3) it preferentially binds selenocysteyl-tRNA(Sec). In contrast to bacterial SelB, however, no binding of MJ0495 protein to the SECIS element of the mRNA was found under the experimental conditions employed which correlates with the fact that MJ0495 lacks the C-terminal domain of the bacterial SelB protein known to bind the SECIS element. It is speculated that in Archaea the functions of bacterial SelB are distributed over at least two proteins, one, serving as the specific translation factor, like MJ0495, and another one, binding to the SECIS which interacts with the ribosome and primes it to decode UGA.

Amino Acid Sequence↗

PubMed1998

Bacterial selenocysteine synthase--structural and functional properties.

Selenocysteine synthase from Escherichia coli is a pyridoxal-5'-phosphate-containing enzyme which catalyses the conversion of seryl-tRNA(Sec) into selenocysteyl-tRNA(Sec). Analysis of amino acid sequences indicated that selenocysteine synthase belongs to the alpha/gamma superfamily of pyridoxal-5'-phosphate-dependent enzymes. To identify the lysine residue carrying the prosthetic group, the genes coding for the selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum were cloned and sequenced and their derived amino acid sequences were aligned with those from E. coli and Haemophilus influenzae. Three lysine residues were found to be conserved; they were mutated into asparagine and one of them, Lys295, was found to be essential for activity. Proteolytic fragmentation of the E. coli enzyme reduced with borohydride, and mass-spectrometric and sequence analysis of the chromophoric peptide proved that Lys295 was modified. Kinetic analysis of the enzyme showed that thiophosphate served as a substrate leading to cysteyl-tRNA(Sec) synthesis, albeit with a 330-fold lower catalytic efficiency. Selenide and, to a much lesser degree, sulfide could also be used by the enzyme but only at much higher concentrations. These data together with the finding that selenophosphate synthetase is highly specific for selenide indicate that the phosphate moiety of selenophosphate provides selenocysteine synthase with the discrimination specificity against sulfur.

Amino Acid Sequence↗

PubMed1998

Functional expression in Escherichia coli of the Haemophilus influenzae gene coding for selenocysteine-containing selenophosphate synthetase.

The selenophosphate synthetases from several organisms contain a selenocysteine residue in their active site where the Escherichia coli enzyme contains a cysteine. The synthesis of these enzymes, therefore, depends on their own reaction product. To analyse how this self-dependence is correlated with the selenium status, e.g. after recovery from severe selenium starvation, we expressed the gene for the selenocysteine-containing selenophosphate synthetase from Haemophilus influenzae (selDHI) in an E. coli DeltaselD strain. Gene selDHI gave rise to a selenium-containing gene product and also supported - via its activity - the formation of E. coli selenoproteins. The results provide evidence either for the suppression of the UGASec codon with the insertion of an amino acid allowing the formation of a functional product or for a bypass of the selenophosphate requirement. We also show that the selenocysteine synthesis and the insertion systems of the two organisms are fully compatible despite conspicuous differences in the mRNA recognition motif.

Bacterial Proteins↗

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

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

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

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↗

PubMed1994

Genes coding for the selenocysteine-inserting tRNA species from Desulfomicrobium baculatum and Clostridium thermoaceticum: structural and evolutionary implications.

The genes (selC) coding for the selenocysteine-inserting tRNA species (tRNA(Sec)) from Clostridium thermoaceticum and Desulfomicrobium baculatum were cloned and sequenced. Although they differ in numerous positions from the sequence of the Escherichia coli selC gene, they were able to complement the selC lesion of an E. coli mutant and to promote selenoprotein formation in the heterologous host. The tRNA(Sec) species from both organisms possess all of the unique primary, secondary, and tertiary structural features exhibited by E. coli tRNA(Sec) (C. Baron, E. Westhof, A. Böck, and R. Giegé, J. Mol. Biol. 231:274-292, 1993). The structural and functional properties of the tRNA(Sec) species from prokaryotes analyzed thus far support the notion that tRNA(Sec) may be an evolutionarily conserved structure whose function in the primordial genetic code was to decode UGA with selenocysteine.

Base Sequence↗