Expression and operon structure of the sel genes of Escherichia coli and identification of a third selenium-containing formate dehydrogenase isoenzyme

Abstract

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 atrobiosis or anaerobiosis nor by the intracellular selenium concentration. The gene encoding the tRNA molecule which is specifically aminoacylated with selenocysteine (seiC) proved to be monocistronic. In contrast, the other three sel genes (seL4, -B, and -D) were shown to be constituents of two unlinked operons. The selA and selB geites formed one transcriptional unit (seL41)7, while selD was shown to be the central gene in an operon including two other genes, the promoter distal of which (topB) encodes topoisomerase HI. The promoter proximal gene (orfl83) 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 orfl83 and topB genes were required neither for selenoprotein biosynthesis nor for selenation of tRNAs. seL4B transcription was driven by a single, weak promoter; however, two major selD operon transcripts were identified. The longer initiated just upstream of the orfl83 gene, whereas the 5' end of the other mapped in a 116-bp nontranslated region between orfl83 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.The products of four genes have been identified as being essential for the incorporation of selenium into macromolecules in Escherichia coli (8,44). The selD gene encodes a protein which is required for incorporation of selenium into both protein and modified tRNAs (28), whereas the products of the other three genes are required for the synthesis of selenopolypeptides (29). The selC gene product is a tRNA (30) which is aminoacylated with serine, and this serine residue is converted to selenocysteine on the tRNA through the joint action of SelD (28) and selenocysteine synthase, the selA gene product ...

Carbon source-dependent expansion of the genetic code in bacteria

“…This regulated recoding allows constitutive formation of Sec-tRNA Sec without disrupting the proteome by reading through UGA codons intended to stop translation. In bacteria and archaea, Sec encoding is not essential for survival, but formate-dependent growth in Methanococcus maripaludis and E. coli requires Sec insertion into formate dehydrogenase (31,32). Fascinatingly, whereas Pylprotein expression in A. arabaticum depends on the presence of TMA, selenoproteins can be overproduced in both bacteria and archaea even when formate is not present in the medium (33).…”

Recognition of the mRNA selenocysteine insertion sequence by the specialized translational elongation factor SELB.

“…The m e c h a n i s m by which SELB participates in selenoprotein synthesis has been analyzed by boundary as well as toeprint experiments (see above), mobilityshift assays (Baron et al 1993b), and genetic analysis (Leinfelder et al 1988bSawers et al 1991). The data suggest that the SELB-GTP-Sec-tRNA sec ternary complex binds within the SECIS and that binding occurs even when the SECIS is saturated with ribosome.…”

“…Selenoproteins typically contain one selenocysteine moiety per peptide chain although the mammalian plasma protein, selenoprotein P, has been reported to have as many as 10 selenocysteine residues (Hill et al 1991(Hill et al , 1993. Other examples of selenoproteins include the three coordinately expressed formate dehydrogenases from Escherichia coli (Berg et al 1991a;Sawers et al 1991) as well as glutathione peroxidase and type I tetraiodothyronine deiodinase from mammals (Forstrom et al 1978;Stadtman 1990;Berry et al 1991). In general, these enzymes catalyze oxidation-reduction reactions, sometimes acting as peroxide scavengers (for recent reviews, see Stadtman 1990;Bock et al 1991;Burk 1991) and share the common feature that the selenocysteine residue has been implicated during catalysis (Wu and Hilvert 1989;Arkowitz and Abeles 1990;Stadtman 1990;Axley et al 1991).…”

Supramolecular organizations in the aerobic respiratory chain of Escherichia coli

“…All activities are fully inhibited by the hemeecopper oxidase inhibitor KCN. The aerobic formate dehydrogenase of E. coli oxidizes formate from the cytoplasm [51] and has been suggested to be able to use O 2 as a final electron acceptor [52]. The co-localization of the 3 enzymes in band 4 and the absence or reduction of the NADH:NBT oxidoreductase activity in the membranes of the E. coli strains devoid of cytochrome bo 3 or bd, respectively, suggests the assembly of a formate oxidase supercomplex, with a stoichiometry of 1:1:1, which is further supported by the estimated mass of band 4, 432 AE 7 kDa, in agreement with the sum of the 3 enzymatic components of the supercomplex hypothesized herein, 446 kDa.…”