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.

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 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 IacZ reporter gene. Efficient read-through ofthe UGA codon, as measured by ,B-galactosidase activity and incorporation of selenium, was dependent on the presence of at least 40 bases offdhF mRNA downstream ofthe UGA codon. There was excellent correlation between the results of the deletion studies and the existence of a putative stem-oop 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 IacZ 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.The incorporation of selenocysteine into selenoproteins is directed by an in-frame UGA codon (for review, see ref. 1). In the case of the selenopolypeptide subunit of formate dehydrogenase H (FDHH) (fdhF gene product) from Escherichia coli it has been shown that the insertion of this nonstandard amino acid into the growing polypeptide chain occurs cotranslationally (2). The incorporation is dependent on the presence of a unique tRNA species (the selC gene product), whose anticodon UCA is complementary to the UGA nonsense codon (3). This tRNA is aminoacylated with L-serine and the L-seryl-tRNAUCA is subsequently converted into selenocysteinyl-tRNAucA by the catalytic action of two proteins, the selA and selD gene products (4, 5). Delivery of selenocysteinyl-tRNAUCA to the ribosome requires a protein (SelB) that specifically binds the charged tRNA (but not seryl-tRNAuCA) and guanine nucleotides and that is considered to act as an elongation factor alternate in its function to elongation factor Tu (6).The present paper deals with the central question as to how the UGA codon ofthefdhF mRNA (UGA140) is discriminated by the ribosome from a chain-termination UGA codon.Evidence is presented that demonstrates that the coding specificity of the UGA140 codon depends on the presence of, minimally, 40 bases of the mRNA immediately downstream of the UGA codon. MATERIALS AND METHODSStrains, Media, and Growth Conditions. The strains of E. coli used in this work are listed in Table 1. The buffered rich medium us...

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.

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 pleiotropicfdh 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. ThefdhB 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 pleiotropicfdh 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 (set) is proposed for pleiotropic fdh mutations affecting selenium metabolism. Four genes have been identified so far: selA and selB (at thefdhA locus), selC (previously fdhC), and selD (previously fdhB).Several enzymes from procaryotic and eucaryotic organisms and a number of tRNA species contain selenium in a covalently bound form (21). For bacteria, the first indication for a biological role of this trace element dates back to 1954, when Pinsent detected that gas production by anaerobic cultures of Escherichia coli depended upon the presence of selenium in the medium (20); enzymological studies revealed that it was the formate dehydrogenase component of the formate-hydrogen-lyase complex which specifically required selenium for activity (14,20). By using [75Se]selenite, it was subsequently demonstrated that the isotope was incorporated into just two proteins in E. coli: an 80-kilodalton polypeptide, which is a constituent component of formate dehydrogenase H (FDHH) and is involved in gas formation, and a 110-kilodalton polypeptide, which is part of formate dehydrogenase N (FDHN) and delivers the electrons from formate to nitrate reductase (8,19). FDHH is formed under anaerobic conditions in the absence of external electron acceptors, whereas synthesis of FDH...

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.

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.

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.