Lambda cI repressor mutants altered in transcriptional activation.
We analysed the in vivo functions of three lambda cI repressor mutants which are phenotypically defective in positive control (pc). For this purpose, we constructed a lambda cI repressor expression system which allows controlled expression of various amounts of lambda cI repressor or its mutants. A five-fold activation of the PRM promoter by wild-type lambda cI repressor is measured in this in vivo system. Two of the pc mutants (pc 1: G43-R and pc 3: E34-K) repress the PRM promoter over a wide range of intracellular concentrations, the lowest being almost identical to the concentration of wild-type lambda cI repressor at which it activates the transcription of its own gene. Only the third pc mutant (pc 2: D38-N) behaves in a manner that would be expected of a true pc mutant, which is unaffected in its DNA binding activity but has lost its activation function. We studied the DNA binding properties of cI repressor and its three pc mutants with a variety of operator constructs in vivo and found that the four repressor proteins differed significantly with respect to their affinities for all operators tested. We also probed the necessity of an acidic residue at position 38 of cI repressor for activation and found that the substitution of aspartic acid 38 by tyrosine does not reduce activation of PRM. Furthermore a substitution with phenylalanine improves the activator function of cI repressor. Our results suggest that amino acid replacements at position 34 or 43 of lambda cI repressor predominantly affect the binding properties of the repressor while some hydrophobic amino acid residues at position 38 are at least as functional in activation as the acidic wild-type amino acid residue.
Kolkhof
P
P
Institut für Genetik, Universität zu Köln, Germany.
Müller-Hill
B
B
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000502:physiology*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins