MSA frequency versus substitution outcome and results from parallel mutagenesisa.

a: “X” = the starting amino acid for LXhX chimeras. “D” = substitution caused widely different outcomes among several chimeras. “A” = Amino acid absent from the YPAL-MSA but allowed repression near or better than parent protein in 2 or more chimeras. “P1” = Amino acid present in the YPAL-MSA but diminished repression below the biologically determined threshold of 13 Miller units for at least one chimera. “P2” = Amino acid present in MSA but mutation diminished repression to the “no repressor” limit (“MIN” in Figure 2) for at least one chimera. “L” = Amino acid was absent in MSA; allows strong repression in LacI though not other chimeras (LacI data are commonly used as a single representative of the family.) “–” = an insufficient number of substitutions were isolated to determine general outcome.b: In the un-mutated chimeras, position 62 differs for each regulatory domain.