Comparing the efficiencies of various non-AUG initiator codons in ALA1. (A) Complementation assays for mitochondrial AlaRS activity. The ala1- strain was transformed with various ALA1 constructs, and the growth phenotypes of the transformants were tested. Complementation of the mitochondrial defect of the ala1- strain was shown by its ability to lose the maintenance plasmid following FOA selection and grow on a YPG plate. The frequency of each non-AUG initiator codon that appeared in the screening is indicated in the parenthesis behind the codon. (B) Assay of initiating activity by Western blots. Upper panel, AlaRS-LexA fusion; lower panel, PGK (as loading controls). (C) Assay of the relative initiating activity by Western blots. Protein extracts prepared from the construct with an ATG initiator codon were 2-fold serially diluted and compared to those from constructs with non-ATG initiator codons. The quantitative data for the relative expression levels of these constructs are shown as a separate diagram at the bottom. (D) RT-PCR. Relative amounts of specific ALA1-lexA mRNAs generated from each construct were determined by RT-PCR. As a control, relative amounts of actin mRNAs were also determined. The ALA1 sequences used in ALA1-lexA constructs 1~11 in (B) were respectively transferred from constructs 1~11 shown in (A). In (C) and (D) the numbers 1~11 (circled) denote constructs shown in (B).