Open AccessNucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli.
Author(s) -
F Zii,
A Birkmann,
Thressa C. Stadtman,
August Böck
Publication year - 1986
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.83.13.4650
Subject(s) - selenocysteine , formate dehydrogenase , stop codon , nucleic acid sequence , open reading frame , biochemistry , coding region , biology , microbiology and biotechnology , peptide sequence , gene , escherichia coli , selenoprotein , amino acid , start codon , chemistry , nucleotide , formate , enzyme , cysteine , glutathione , glutathione peroxidase , catalysis
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.