Rheostat, toggle-like, and neutral behaviors of nonconserved linker positions^a.

a: “R, rheostatic (progressive) changes that span >2 orders of magnitude; “r”, rheostat character but <2 orders of magnitude. “T”, toggle-like. “N”, neutral (within 2-fold change); “n”, between 2 and ∼5-fold change. “#”, insufficient number of substitutions to determine behavior. “–”, not mutated. “x”, weak or no measureable repression for any substitution. b:. Substitutions generated 3 states instead of a continuum. Either 2- or 3-state toggles might reveal rheostat behavior if additional amino acid substitutions were characterized. However, in addition to reported variants, no intermediates were identified for the 2-state toggles during colony selection, which involved visual inspection of lac repression for several hundred bacterial colonies expressing randomly mutated chimeras. c: In designating a neutral position, we invoked the caveat “most amino acids” because, for example, proline and glycine substitutions can have large backbone effects. Nevertheless, all reported variants bound DNA in the pull-down assay, which indicated that the protein structure was not grossly distorted. d: Seven (7) of 11 amino acids are neutral; the remaining 4 have rheostat character. e: A subset of positions had rheostat behavior, and another subset abolished detectable repression. f: Substitution results were between neutral and rheostat behavior (∼5–9 fold change). g: Four substitutions convey equally enhanced repression; another 6 have rheostat character.