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A detailed analysis of the expression of the sel genes, the products of which are necessary for the specific incorporation of selenium into macromolecules in Escherichia coli, showed that transcription was constitutive, being influenced neither by aerobiosis or anaerobiosis nor by the intracellular selenium concentration. The gene encoding the tRNA molecule which is specifically aminoacylated with selenocysteine (selC) proved to be monocistronic. In contrast, the other three sel genes (selA, -B, and -D) were shown to be constituents of two unlinked operons. The selA and selB genes formed one transcriptional unit (sel vector AB), while selD was shown to be the central gene in an operon including two other genes, the promoter distal of which (topB) encodes topoisomerase III. The promoter proximal gene (orf183) was sequenced and shown to encode a protein consisting of 183 amino acids (Mr, 20,059), the amino acid sequence of which revealed no similarity to any currently known protein. The products of the orf183 and topB genes were required neither for selenoprotein biosynthesis nor for selenation of tRNAs. selAB transcription was driven by a single, weak promoter; however, two major selD operon transcripts were identified. The longer initiated just upstream of the orf183 gene, whereas the 5' end of the other mapped in a 116-bp nontranslated region between orf183 and selD. Aerobic synthesis of all four sel gene products incited a reexamination of a weak 110-kDa selenopolypeptide which is produced under these conditions. The aerobic appearance of this 110-kDa selenopolypeptide was not a consequence of residual expression of the gene encoding the 110-kDa selenopolypeptide of the anaerobically inducible formate dehydrogenase N (FDHN) enzyme, as previously surmised, but rather resulted from the expression of a gene encoding a third, distinct selenopolypeptide in E. coli. A mutant strain no longer capable of synthesizing the 80- and 110-kDa selenopolypeptides of FDHH and FDHN, respectively, still synthesized this alternative 110-kDa selenopolypeptide which was present at equivalent levels in cells grown aerobically and anaerobically with nitrate. Furthermore, this strain exhibited a formate- and sel gene-dependent respiratory activity, indicating that it is probable that this selenopolypeptide constitutes a major component of the formate oxidase, an enzyme activity initially discovered in aerobically grown E. coli more than 30 years ago.