(A) Schematic of inserts in +1 frame in GLN4(1-99)-GFP reporters used to identify the contributions of individual CGA-CGG pairs to frameshifting. Sequences with all possible combinations of zero, one, two or three inhibitory CGA-CGG pairs (I, shown in cyan) [substituting the synonymous optimal pair AGA-AGA (O, shown in orange) at other positions] were assayed. (B) All constructs with an inhibitory codon pair at the first position (III, IIO, IOI, IOO) showed high levels of frameshifting in all three mutants. Analysis of effects of asc1Δ, mbf1Δ and asc1Δ mbf1Δ mutations on expression of GLN4(1-99)-GFP reporters with the indicated position and number of inhibitory codon pairs. (C, D) Analysis of effects of asc1Δ, mbf1Δ and asc1Δ mbf1Δ mutations on expression of GLN4(1-99)-GFP reporters containing three Arg-Arg codon pairs (AGA-AGA versus CGA-CGG) in 0, +1, and −1 reading frames. In (C), the first codon pair is followed by CAC, while in (D) the first codon pair is followed by TTC. (E) Analysis of effects of native yeast gene sequences containing a single CGA-CGG codon pair on in-frame and frameshifted expression of GFP. In each case, six codons upstream and downstream of the CGA-CGG were inserted into the GLN4(1-99)-GFP reporter in-frame and with a +1 frameshift after the inserted sequence. Expression of GFP/RFP was measured in wild type, asc1Δ, mbf1Δ and asc1Δ mbf1Δ mutants.