RESUMEN
Gene grdA, which encodes selenoprotein A of the glycine reductase complex from Clostridium sticklandii, was identified and characterized. This gene encodes a protein of 158 amino acids with a calculated M(r) of 17,142. The known sequence of 15 amino acids around the selenocysteine residue and the known carboxy terminus of the protein are correctly predicted by the nucleotide sequence. An opal termination codon (TGA) corresponding to the location of the single selenocysteine residue in the polypeptide was found in frame at position 130. The C. sticklandii grdA gene was inserted behind the tac promotor of an Escherichia coli expression vector. An E. coli strain transformed with this vector produced an 18-kDa polypeptide that was not detected in extracts of nontransformed cells. Affinity-purified anti-C. sticklandii selenoprotein A immunoglobulin G reacted specifically with this polypeptide, which was indistinguishable from authentic C. sticklandii selenoprotein A by immunological analysis. Addition of the purified expressed protein to glycine reductase protein components B and C reconstituted the active glycine reductase complex. Although synthesis of enzymically active protein A depended on the presence of selenium in the growth medium, formation of immunologically reactive protein did not. Moreover, synthesis of enzymically active protein in a transformed E. coli selD mutant strain indicated that there is a nonspecific mechanism of selenocysteine incorporation. These findings imply that mRNA secondary structures of C. sticklandii grdA are not functional for UGA-directed selenocysteine insertion in the E. coli expression system.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Proteínas Bacterianas/genética , Clostridium/enzimología , Clostridium/genética , Escherichia coli/genética , Genes Bacterianos , Complejos Multienzimáticos/genética , Selenio/metabolismo , Aminoácido Oxidorreductasas/aislamiento & purificación , Aminoácido Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Complejo Antígeno-Anticuerpo , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Codón/genética , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Cinética , Datos de Secuencia Molecular , Peso Molecular , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Conformación de Ácido Nucleico , Plásmidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Selenoproteínas , TermodinámicaAsunto(s)
Aminoácido Oxidorreductasas/genética , Proteínas Bacterianas/genética , Cisteína/análogos & derivados , Genes Bacterianos , Complejos Multienzimáticos/genética , Selenio , Secuencia de Bases , Clostridium/enzimología , Cisteína/genética , Datos de Secuencia Molecular , Selenocisteína , SelenoproteínasRESUMEN
The gene encoding the selenoprotein A component of glycine reductase was isolated from Clostridium purinolyticum. The nucleotide sequence of this gene (grdA) was determined. The opal termination codon (TGA) was found in-frame at the position corresponding to the location of the selenocysteine residue in the gene product. A comparison of the nucleotide sequences and secondary mRNA structures corresponding to the selenoprotein A gene and the fdhF gene of Escherichia coli formate dehydrogenase shows that there is a similar potential for regulation of the specific insertion of selenocysteine at the UGA codon.