We have isolated same-site and second-site revertants that restore partial activity, wild-type activity, or greater than wild-type activity, to λ repressor proteins bearing different mutations in the DNA binding domain. In some cases the revertant repressors contain same-site substitutions that are similar to the wild-type side-chain (e.g. Tyr22 → Phe, Ser77 → Thr). The activity of these revertants makes it possible to assess the role of specific hydrogen bonds and/or packing interactions in repressor structure and function. In other same-site revertants, a very different type of residue is introduced (e.g. Ser35 → Leu, Gly48 → Asn). This indicates that the chemical and steric requirements at these side-chain positions are relaxed. Two of the second-site revertants, Glu34 → Lys and Gly48 → Ser, restore activity to more than one primary mutant. Both substitutions apparently increase the affinity of the repressor-operator interaction by introducing new contacts with operator DNA. These results suggest that reversion may be a generally applicable method for identifying sequence changes that increase the activity of a protein to greater than wild-type levels.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Structural Biology