The activation defect of a λc I positive control mutant

Abstract

“Positive control” mutants of the c I protein of bacteriophage λ (λc I) bind DNA but, unlike the wild-type protein, fail to activate transcription. According to the original interpretation of Ptashne and co-workers, these mutants bear amino acid substitutions that disrupt a stimulatory interaction between λc I bound at operator site O R2 and RNA polymerase bound at promoter P RM, an idea supported by kinetic analysis in one case. Genetic analysis has suggested that one residue in particular, glutamate 34 (E34), is critical for the stimulatory effect of wild-type λc I. More recently, however, Kolkhof and Müller-Hill have challenged this view, suggesting that mutant E34K fails to activate because it binds at unusually low concentrations to O R3, a site that mediates repression of P RM. To test this hypothesis, we have examined the behaviour of the λc I-E34K mutant both in vitro and in vivo by assaying transcription from P RM and monitoring operator site occupancy over a range of protein concentrations. Our results are inconsistent with the interpretation of Kolkhof and Müller-Hill, and demonstrate that under conditions where λ operator O R2 is fully occupied and operator O R3 is vacant, wild-type λc I activates transcription from promoter P RM whereas the mutant does not.