Barriers to heterologous expression of a selenoprotein gene in bacteria.
The specificity parameters counteracting the heterologous expression in Escherichia coli of the Desulfomicrobium baculatum gene (hydV) coding for the large subunit of the periplasmic hydrogenase which is a selenoprotein have been studied. hydV'-'lacZ fusions were constructed, and it was shown that they do not direct the incorporation of selenocysteine in E. coli. Rather, the UGA codon is efficiently suppressed by some other aminoacyl-tRNA in an E. coli strain possessing a ribosomal ambiguity mutation. The suppression is decreased by the strA1 allele, indicating that the hydV selenocysteine UGA codon has the properties of a "normal" and suppressible nonsense codon. The SelB protein from D. baculatum was purified; in gel shift experiments, D. baculatum SelB displayed a lower affinity for the E. coli fdhF selenoprotein mRNA than E. coli SelB did and vice versa. Coexpression of the hydV'-'lacZ fusion and of the selB and tRNA(Sec) genes from D. baculatum, however, did not lead to selenocysteine insertion into the protein, although the formation of the quaternary complex between SelB, selenocysteyl-tRNA(Sec), and the hydV mRNA recognition sequence took place. The results demonstrate (i) that the selenocysteine-specific UGA codon is readily suppressed under conditions where the homologous SelB protein is absent and (ii) that apart from the specificity of the SelB-mRNA interaction, a structural compatibility of the quaternary complex with the ribosome is required.
Tormay
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P
Lehrstuhl für Mikrobiologie der Universität München, Munich, Germany.
Böck
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A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003062:Codon; D003901:Desulfovibrio / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006864:Hydrogenase / Q000096:biosynthesis* / Q000235:genetics; D008667:Metalloproteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D017279:Selenocysteine / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D013489:Suppression, Genetic
D001426:Bacterial Proteins; D003062:Codon; D008667:Metalloproteins; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; C061878:SelB protein, Bacteria; C112705:selenocysteinyl-tRNA; D017279:Selenocysteine; D006864:Hydrogenase