Targeted insertion of selenocysteine into the alpha subunit of formate dehydrogenase from Methanobacterium formicicum

Abstract

Selenocysteine incorporation into proteins is directed by an opal (UGA) codon and requires the existence of a stem-loop structure in the mRNA flanking the UGA at its 3' side. To analyze the sequence and secondary-structure requirements for UGA decoding, we have introduced mutations into the fdhA gene from Methanobacterium formicicum, which codes for the alpha subunit of the F420-reducing formate dehydrogenase. The M. formicicum enzyme contains a cysteine residue at the position where the Escherichia coli formate dehydrogenase H carries a selenocysteine moiety. The codon (UGC) for this cysteine residue was changed into a UGA codon, and mutations were successively introduced at the 5' and 3' sides to generate a stable secondary structure of the mRNA and to approximate the sequence of the predicted E. coli fdhF mRNA hairpin structure. It was found that introduction of the UGA and generation of a stable putative stem-loop structure were not sufficient for decoding with selenocysteine. Efficient selenocysteine incorporation, however, was obtained when the loop and the immediately adjacent portion of the putative stem had a sequence identical to that present in the E. coli fdhF mRNA structure.