The gene (fdhF) coding for the selenopolypeptide of the benzylviologen-linked formate dehydrogenase of Escherichia coli was cloned and its nucleotide sequence was determined. ThefdhF 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 ,B-galactosidase fusion proteins were synthesized when the deletion clones or gene fusions, respectively, contained thefdhF 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-terminalfdhF part contained the UGA codon and was independent of the presence of selenium when a more upstream part offdhF was fused to lacZ. The results are consistent with a co-translational selenocysteine incorporation mechanism.

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 Nterminal parts offdhF 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 anfdhF 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 inactivefdhF 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.

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.

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The fdhF gene, encoding the selenopolypeptide of formate dehydrogenase (FDHH), has a -12/-24 nif-type consensus promoter. A cis-acting DNA element, which is required for the regulation of the promoter by formate under anaerobic conditions, has been identified. This regulatory sequence of about 25 bp in length is located 110 bp upstream of the transcription start site. By analysing a variety of mutant constructs in this region (5' deletions, internal deletions and point mutations) we were able to identify a hexanucleotide sequence -GTCACG-, which is important for the formate regulation of the fdhF promoter. The data also indicate that this element has many of the properties characteristic of eukaryotic enhancers.