The basis for the super-repressor phenotypes of the AV77 and EK18 mutants of trp repressor

The DNA-binding properties of two super-repressor mutants of the Escherichi a coli frp repressor, EK18 and AV77, have been investigated using steady-st ate fluorescence anisotropy measurements, in order to further elucidate the basis for their super-repressor phenotypes. Several suggestions have been previously proposed as the basis for the super-repressor phenotype of EK18 and AV77. For the negative to positive charge change EK18 mutant, increased electrostatic interactions between the EK18 mutant and the operator and in creased protein-protein interactions between EK18 dimers have been suggeste d as contributing to the super-repressor phenotype of this mutant. We show that EK18 dimers actually bind to wild-type and variant operator sequences with a decrease in apparent cooperativity and an increase in affinity, comp ared to WTTR dimers. Thus, the EK18 super-repressor phenotype is not due to increased cooperative binding between EK18 dimers. These results support t he hypothesis that the super-repressor phenotype of EK18 arises from increa sed electrostatic interactions between the mutant and DNA. in the case of t he AV77 mutant, weaker binding affinity of apo-AV77 to non-specific DNA, in creased selectivity of binding of AV77 for the operator, and a higher popul ation of folded functional AV77 dimers available to bind the operator under limiting L-Trp conditions in vivo, have been proposed for the super-repres sor phenotype of this mutant. We show that like the EK18 mutant, apoAV77 bi nds with higher affinity to nonspecific DNA compared to apo-WTTR and that t he holo-AV77 mutant does not bind with higher selectivity to the operator, has had been previously proposed. We therefore conclude that the super-repr essor phenotype of the AV77 mutant is due to an increase in the population of folded, functional AV77 dimers, under limiting L-Trp conditions if vivo. (C) 2000 Academic Press.