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 biological requirement of the trace element selenium was recognized 40 years ago. Selenium is incorporated into several enzymes and transfer RNA species of both prokaryotic and eukaryotic origin. In enzymes which contain a selenopolypeptide, selenium is present as covalently bound selenocysteine which participates in the catalytic reaction. Sequence analysis of the genes coding for two selenoproteins, formate dehydrogenase H from Escherichia coli and glutathione peroxidase from mouse and man, demonstrated that an in-frame UGA opal nonsense codon directs the incorporation of selenocysteine. In the case of formate dehydrogenase incorporation occurs cotranslationally. Recently, we identified four genes whose products are required for selenocysteine incorporation in E. coli. We report here that one of these genes codes for a tRNA species with unique properties. It possesses an anticodon complementary to UGA and deviates in several positions from sequences, until now, considered invariant in all tRNA species. This tRNA is aminoacylated with L-serine by the seryl-tRNA ligase which also charges cognate tRNASer. Selenocysteine, therefore, is synthesized from a serine residue bound to a natural suppressor tRNA which recognizes UGA.
During the biosynthesis of selenoproteins in both prokaryotes and eukaryotes, selenocysteine is cotranslationally incorporated into the nascent polypeptide chain through a process directed by a UGA codon that normally functions as a stop codon. Recently, four genes have been identified whose products are required for selenocysteine incorporation in Escherichia coli. One of these genes, selC, codes for a novel transfer RNA species (tRNAUCA) that accepts serine and cotranslationally inserts selenocysteine by recognizing the specific UGA codon. The serine residue attached to this tRNA is converted to selenocysteine in a reaction dependent on functional selA and selD gene products. By contrast, the selB gene product (SELB) is not required until after selenocysteyl-tRNA biosynthesis. Here we present evidence indicating that SELB is a novel translation factor. The deduced amino-acid sequence of SELB exhibits extensive homology with the sequences of the translation initiation factor-2 (IF-2) and elongation factor Tu (EF-Tu). Furthermore, purified SELB protein binds guanine nucleotides in a 1:1 molar ratio and specifically complexes selenocysteyl-tRNAUCA, but does not interact with seryl-tRNAUCA. Thus, SELB could be an amino acid-specific elongation factor, replacing EF-Tu in a special translational step.
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...
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