Recoding UAG to selenocysteine in Saccharomyces cerevisiae

Sec-specific Gal4 reporter. (A) Design of Gal4 reporter with up to two Cys coded for by an Am codon (C11Am and/or C21Am) in the DNA binding domain. If Ser is inserted in either of those positions, an inactive Gal4 will be expressed which is unable to bind to the Gal4 upstream activation sequence (UAS) to activate transcription of URA3. The same is true if suppression does not occur and Gal4 is truncated. URA3 promotes S. cerevisiae growth in the absence of uracil. If Sec is inserted, then the DNA binding domain of Gal4 is active to bind to the GAL4_UAS and activate URA3 transcription. (B) S. cerevisiae growth is dependent on the presence of a Cys (or Sec) at positions 11 and 21 to produce active Gal4. Mutation of either of those positions to Ser (C11S or C21S) does not promote cell growth. (C) Testing individual (C11Am or C21Am) or dual (C11Am/C21Am) Sec insertion found that SctRNA^Sec_CUA-3 promotes cell growth better than SctRNA^Sec_CUA-1.