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SummaryGreat 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, tRNA^"'' 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.

In-frame deletions were introduced into each of the eight genes of the hyc operon coding for products required for the formation of the formate hydrogenlyase (FHL) system. The deletions were transferred to the chromosome and the resulting mutants were analysed for development of formate dehydrogenase H and hydrogenase 1, 2 and 3 activity. It was found that hycA, the promoter-proximal gene, is a regulatory gene and that it codes for a product counteracting transcriptional activation by FhlA. Deletions within the hycB to hycH genes specifically affected formate dehydrogenase H activity or hydrogenase 3 activity, or both. None of the mutations affected hydrogenase 1 or 2 activity. A model is proposed for the functional interaction of the different hyc operon gene products in the formate hydrogenlyase complex, which is based on the results of the mutational analysis, on the determination of the subcellular localization of the FdhF, HycE, HycF and HycG polypeptides and on the similarity of hyc gene product sequences with those from other hydrogenase systems. HycH, the product of the most promoter-distal gene, does not seem to form part of the functional FHL complex but rather is required for the conversion of a precursor form of the large subunit of hydrogenase 3 into the mature form.

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.

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