RESUMO
A 3.3 kb HindIII genomic DNA fragment from Clostridium pasteurianum ATCC 6013 which hybridized to the anfDGK genes for the Fe-only 'alternative' nitrogenase from Azotobacter vinelandii was cloned. Open reading frames (ORFs D, G, and K) with high sequence identity to anfD, anfG, and part of anfK were located in the nucleotide sequence obtained for 2494 bp of this fragment. In C. pasteurianum, ORFD maps approximately 1.8 kb downstream of nifH3 and is transcribed in the same direction. There was no evidence for additional copies of ORFDGK-like sequences in the genome of C. pasteurianum, other than those encoding the Mo-nitrogenase. Physiological and biochemical studies suggest that a nitrogenase not requiring molybdenum may occur in C. pasteurianum. This enzyme is probably encoded by nifH3 and ORFs D, G, and K identified here.
Assuntos
Clostridium/genética , Nitrogenase/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Nitrogenase/química , Fases de Leitura AbertaRESUMO
Great excitement was elicited in the field of selenium biochemistry in 1986 by the parallel discoveries that the genes encoding the selenoproteins glutathione peroxidase and bacterial formate dehydrogenase each contain an in-frame TGA codon within their coding sequence. We now know that this codon directs the incorporation of selenium, in the form of selenocysteine, into these proteins. Working with the bacterial system has led to a rapid increase in our knowledge of selenocysteine biosynthesis and to the exciting discovery that this system can now be regarded as an expansion of the genetic code. The prerequisites for such a definition are co-translational insertion into the polypeptide chain and the occurrence of a tRNA molecule which carries selenocysteine. Both of these criteria are fulfilled and, moreover, tRNASec even has its own special translation factor which delivers it to the translating ribosome. It is the aim of this article to review the events leading to the elucidation of selenocysteine as being the 21st amino acid.
Assuntos
Cisteína/análogos & derivados , Selênio/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Evolução Biológica , Códon , Cisteína/biossíntese , Cisteína/genética , Cisteína/metabolismo , Dados de Sequência Molecular , RNA de Transferência/genética , SelenocisteínaRESUMO
The formate dehydrogenase (FDHF) of Escherichia coli is a selenocysteine-containing protein that occurs as a component of the formate-hydrogen lyase complex. The gene encoding this 80 kd polypeptide contains a TGA codon in the open reading frame. Several indirect lines of evidence showed earlier that the selenocysteine residue in the protein is inserted co-translationally in a TGA (UGA) dependent process. Direct proof that the selenocysteine is present in the polypeptide in the position corresponding to TGA as predicted from the gene sequence was obtained by automated amino acid sequence analysis of a 75Se-containing peptide isolated from the protein. Construction of a fusion gene comprising a small segment of the fdhF gene linked to the lacZ gene as reporter greatly facilitated isolation of the selenocysteine-containing protein. Subsequent cleavage of this isolated gene product with endoproteinase Asp-N gave rise to an easily purified small selenocysteine-containing peptide that was amenable to amino acid sequence analysis.
Assuntos
Códon/genética , Cisteína/análogos & derivados , Escherichia coli/enzimologia , Formiato Desidrogenases/genética , Genes Bacterianos/genética , Sequência de Aminoácidos , Aminoácidos/análise , Sequência de Bases , Cromatografia Líquida , Cisteína/genética , Endopeptidases , Escherichia coli/genética , Formiato Desidrogenases/química , Metaloendopeptidases , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Selênio , Selenocisteína , beta-Galactosidase/metabolismoRESUMO
The fdhF gene encoding the 80-kDa selenopolypeptide subunit of formate dehydrogenase H from Escherichia coli contains an in-frame TGA codon at amino acid position 140, which encodes selenocysteine. We have analyzed how this UGA "sense codon" is discriminated from a UGA codon signaling polypeptide chain termination. Deletions were introduced from the 3' side into the fdhF gene and the truncated 5' segments were fused in-frame to the lacZ reporter gene. Efficient read-through of the UGA codon, as measured by beta-galactosidase activity and incorporation of selenium, was dependent on the presence of at least 40 bases of fdhF mRNA downstream of the UGA codon. There was excellent correlation between the results of the deletion studies and the existence of a putative stem-loop structure lying immediately downstream of the UGA in that deletions extending into the helix drastically reduced UGA translation. Similar secondary structures can be formed in the mRNAs coding for other selenoproteins. Selenocysteine insertion cartridges were synthesized that contained this hairpin structure and variable portions of the fdhF gene upstream of the UGA codon and inserted into the lacZ gene. Expression studies showed that upstream sequences were not required for selenocysteine insertion but that they may be involved in modulating the efficiency of read-through. Translation of the UGA codon was found to occur with high fidelity since it was refractory to ribosomal mutations affecting proofreading and to suppression by the sup-9 gene product.
Assuntos
Aldeído Oxirredutases/genética , Códon/genética , Cisteína/análogos & derivados , Escherichia coli/genética , Formiato Desidrogenases/genética , RNA Mensageiro/genética , Selênio/metabolismo , Sequência de Bases , Clonagem Molecular , Cisteína/metabolismo , DNA Recombinante/metabolismo , Escherichia coli/enzimologia , Expressão Gênica , Genótipo , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos , Mapeamento por Restrição , Radioisótopos de Selênio , Selenocisteína , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Mutants of Escherichia coli were isolated which were affected in the formation of both formate dehydrogenase N (phenazine methosulfate reducing) (FDHN) and formate dehydrogenase H (benzylviologen reducing) (FDHH). They were analyzed, together with previously characterized pleiotropic fdh mutants (fdhA, fdhB, and fdhC), for their ability to incorporate selenium into the selenopolypeptide subunits of FDHN and FDHH. Eight of the isolated strains, along with the fdhA and fdhC mutants, maintained the ability to selenylate tRNA, but were unable to insert selenocysteine into the two selenopolypeptides. The fdhB mutant tested had lost the ability to incorporate selenium into both protein and tRNA. fdhF, which is the gene coding for the 80-kilodalton selenopolypeptide of FDHH, was expressed from the T7 promoter-polymerase system in the pleiotropic fdh mutants. A truncated polypeptide of 15 kilodaltons was formed; but no full-length (80-kilodalton) gene product was detected, indicating that translation terminates at the UGA codon directing the insertion of selenocysteine. A mutant fdhF gene in which the UGA was changed to UCA expressed the 80-kilodalton gene product exclusively. This strongly supports the notion that the pleiotropic fdh mutants analyzed possess a lesion in the gene(s) encoding the biosynthesis or the incorporation of selenocysteine. The gene complementing the defect in one of the isolated mutants was cloned from a cosmid library. Subclones were tested for complementation of other pleiotropic fdh mutants. The results revealed that the mutations in the eight isolates fell into two complementation groups, one of them containing the fdhA mutation. fdhB, fdhC, and two of the new fdh isolates do not belong to these complementation groups. A new nomenclature (sel) is proposed for pleiotropic fdh mutations affecting selenium metabolism. Four genes have been identified so far: selA and selB (at the fdhA locus), selC (previously fdhC), and selD (previously fdhB).
Assuntos
Aldeído Oxirredutases/genética , Escherichia coli/genética , Formiato Desidrogenases/genética , Genes Bacterianos , Selênio/metabolismo , Clonagem Molecular , Cisteína/análogos & derivados , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Teste de Complementação Genética , Mutação , Plasmídeos , Regiões Promotoras Genéticas , SelenocisteínaRESUMO
The regulatory elements involved in expression of the gene (fdhF) for the selenopolypeptide of formate dehydrogenase and of a gene (or transcriptional unit) (hyd) specifically responsible for the formation of the gas-evolving hydrogenase (hydrogenase 3) in Escherichia coli were investigated. Formate (or a product of it) is required for expression of both systems since in a pyruvate-formate-lyase deficient mutant induction occurs only when formate is supplemented externally. Under this condition, formate can partially overcome repression by nitrate. The transcription of both the fdhF gene and the hydrogenase-3-encoding systems is independent of the presence of a wild-type fnr gene when formate is present, supporting the view that the Fnr effect on the formation of the formate-hydrogen-lyase pathway is indirect. Mutations blocking the synthesis of a functional molybdenum cofactor also had no major affect on fdhF and hyd expression. The nucleotide sequence of the 5' flanking region of the fdhF gene was determined and the transcription start point of the fdhF gene was localized by nuclease S1 mapping. Nuclease Bal31 generated deletion clones were constructed and the regulation of their expression was studied. Anaerobic expression and induction by formate depended on the presence of a stretch of approximately 185 nucleotides upstream of the translation start. Elements mediating formate induction and oxygen or nitrate repression could not be separated physically. The regulatory features of the fdhF upstream region bear striking resemblance to systems whose expression are dependent upon upstream activating elements.
Assuntos
Aldeído Oxirredutases/genética , Escherichia coli/genética , Formiato Desidrogenases/genética , Genes Bacterianos , Hidrogenase/genética , Transcrição Gênica , Anaerobiose , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/análise , DNA Recombinante , Endodesoxirribonucleases , Escherichia coli/enzimologia , Regulação da Expressão Gênica , Imunoeletroforese , Mutação , Hibridização de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Bacteriano/análise , Transdução GenéticaRESUMO
The structural gene (fdhF) for the 80-kDa selenopolypeptide of formate dehydrogenase (formate:benzyl viologen oxidoreductase, EC 1.2.--.--) from Escherichia coli contains an in-frame UGA codon at amino acid position 140 that is translated. Translation of gene fusions between N-terminal parts of fdhF with lacZ depends on the availability of selenium in the medium when the hybrid gene contains the UGA codon; it is independent of the presence of selenium when an fdhF portion upstream of the UGA position is fused to lacZ. Transcription does not require the presence of selenium in either case. By localized mutagenesis, the UGA codon was converted into serine (UCA) and cysteine (UGC and UGU) codons. Each mutation relieved the selenium dependency of fdhF mRNA translation. Selenium incorporation was completely abolished in the case of the UCA insertion and was reduced to about 10% when the UGA was replaced by a cysteine codon. Insertion of UCA yielded an inactive fdhF gene product, while insertion of UGC and UGU resulted in polypeptides with lowered activities as components in the system formerly known as formate hydrogenlyase. Altogether the results indicate that the UGA codon at position 140 directs the cotranslational insertion of selenocysteine into the fdhF polypeptide chain.
Assuntos
Aldeído Oxirredutases/genética , Códon , Cisteína/análogos & derivados , Escherichia coli/genética , Formiato Desidrogenases/genética , Genes Bacterianos , Genes , Mutação , Biossíntese de Proteínas , RNA Mensageiro , Selênio/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cisteína/metabolismo , Enzimas de Restrição do DNA , Escherichia coli/enzimologia , Hibridização de Ácido Nucleico , Plasmídeos , Ácido Selenioso , Selênio/farmacologia , SelenocisteínaRESUMO
The gene (fdhF) coding for the selenopolypeptide of the benzylviologen-linked formate dehydrogenase of Escherichia coli was cloned and its nucleotide sequence was determined. The fdhF gene contains, within an open reading frame coding for a protein of 715 amino acids (calculated molecular weight, 79,087), an opal (UGA) nonsense codon in amino acid position 140. Existence of this nonsense codon was confirmed by physical recloning and resequencing. Internal and terminal deletion clones and lacZ fusions of different N-terminal parts of fdhF were constructed and analyzed for selenium incorporation. Selenylated truncated polypeptide chains or beta-galactosidase fusion proteins were synthesized when the deletion clones or gene fusions, respectively, contained the fdhF gene fragment coding for the selenopolypeptide sequence from amino acid residue 129 to amino acid residue 268. Translation of the lacZ part of the fusions required the presence of selenium in the medium when the N-terminal fdhF part contained the UGA codon and was independent of the presence of selenium when a more upstream part of fdhF was fused to lacZ. The results are consistent with a co-translational selenocysteine incorporation mechanism.
Assuntos
Aldeído Oxirredutases/genética , Cisteína/análogos & derivados , Escherichia coli/genética , Formiato Desidrogenases/genética , Selênio/metabolismo , Sequência de Bases , Clonagem Molecular , Códon , Cisteína/metabolismo , Escherichia coli/enzimologia , Regulação da Expressão Gênica , Genes Bacterianos , Peso Molecular , Biossíntese de Proteínas , Selenocisteína , Supressão GenéticaRESUMO
The site of integration of phage M mu d (Ap lac) in mutant M9s which leads to deficiency of formic dehydrogenase (benzylviologen-linked) activity was determined. It was shown that the phage had inserted into the gene for the seleno-polypeptide of the enzyme (80 kd) leading to the formation of a truncated peptide (60 kd) still able to incorporate Se. Synthesis of the truncated polypeptide is subject to the same regulatory signals as that of the wild-type enzyme. The formation of the 110 kd seleno-polypeptide, which is a constituent component of the formic dehydrogenase from the formate-nitrate respiratory pathway, is unimpaired in mutant M9s. The location of the gene for the 80 kd seleno-polypeptide was mapped at 92.4 min of the Escherichia coli chromosome.
Assuntos
Aldeído Oxirredutases/genética , Cromossomos Bacterianos , Escherichia coli/genética , Formiato Desidrogenases/genética , Autorradiografia , Bacteriófago mu/genética , Mapeamento Cromossômico , Escherichia coli/enzimologia , Formiato Desidrogenases/análise , Genes Bacterianos , Marcadores Genéticos , Peptídeos/genética , SelênioRESUMO
The regulation of synthesis of the hydrogenase which is a component of the formate hydrogen-lyase complex was studied by means of a strain of Escherichia coli possessing a transcriptional fusion of the hydrogenase gene (hyd) with the lacZ gene (hyd::lac fusion). Formation of active hydrogenase in the wild strain requires the presence of nickel in the medium; transcription of the hyd gene, however, is independent from the presence of Ni2+. Ni2+ addition to Ni2+-prestarved cells did not lead to any activation of presumptive hydrogenase apoprotein. Regulatory mutants were isolated in which nitrate repression of hyd::lac expression was relieved. Two main classes of regulatory mutants were identified: (i) Mutants with a defect in nitrate reductase; (ii) mutants with a cis-dominant regulatory mutation closely linked to the hyd::lac fusion. In the presence of formate which acts as an inducer, the hyd::lac fusion was also expressed under aerobic conditions. The results infer that nitrate repression of transcription of the hydrogenase structural gene is not effected by nitrate itself but requires the function of the electron transport chain leading to nitrate and that mutations in the promoter/operator region of the hyd cistron may confer insensitivity to redox control both by oxygen and nitrate.
Assuntos
Aldeído Oxirredutases/biossíntese , Escherichia coli/enzimologia , Formiato Desidrogenases/biossíntese , Escherichia coli/genética , Mutação , Níquel/farmacologia , Nitratos/farmacologia , Transdução Genética , beta-Galactosidase/genéticaRESUMO
Mutants of Escherichia coli were isolated in which transcription of the structural genes for hydrogenase (hyd) and for one of the components of formate dehydrogenase (fdh) (of the formate hydrogen-lyase complex) is coupled with that of the lacZ gene. They were--together with lac fusions of the nifH and nifL genes from Klebsiella--used to study regulation by redox control, of the expression of the respective structural genes. The following results were obtained: (i) beta-galactosidase synthesis was fully repressed in the presence of O2 or nitrate (anaerobically), and induced in the absence of an external electron acceptor. Fumarate as terminal electron acceptor only marginally affected nif expression and partially repressed hyd and fdh expression. Redox control of the synthesis of hydrogenase and formate dehydrogenase, therefore, (as well as that of nif) acts at the level of transcription; the size of the redox potential seems to be correlated with the amount of repression; (ii) beta-galactosidase synthesis in the hyd:: lac and fdh::lac fusion strains is induced by formate. At high concentrations formate reverses the repression by nitrate and fumarate but not that by oxygen.