The activation defect of a lambda cI positive control mutant.
"Positive control" mutants of the cI protein of bacteriophage lambda (lambda cI) 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 lambda cI bound at operator site O(R)2 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 lambda cI. More recently, however, Kolkhof and Muller-Hill have challenged this view, suggesting that mutant E34K fails to activate because it binds at unusually low concentrations to O(R)3, a site that mediates repression of P(RM). To test this hypothesis, we have examined the behaviour of the lambda cI-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 Muller-Hill, and demonstrate that under conditions where lambda operator O(R)2 is fully occupied and operator O(R)3 is vacant, wild-type lambda cI activates transcription from promoter P(RM) whereas the mutant does not.
Whipple
F W
FW
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Ptashne
M
M
Hochschild
A
A
D010582:Bacteriophage lambda / Q000235:genetics / Q000276:immunology; D003429:Cross Reactions; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015533:Transcriptional Activation; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D007544:Isopropyl Thiogalactoside; D003850:Deoxyribonuclease I