Mutants with substitutions for Glu171 in the catabolite activator protein (CAP) of Escherichia coli activate transcription from the lac promoter.
Single amino acid substitutions for residue Glu171 in helix E of the catabolite gene activator protein (CAP) of Escherichia coli have been reported to abolish activation of transcription without impairing binding to the CAP site of the lac promoter. The negative charge of Glu171 was proposed to transmit the activating signal from CAP to RNA polymerase. However, this idea has been challenged by later work. We set up a system to re-examine this issue. We analysed the ability of mutant CAP-E171L and CAP-E171K proteins to bind a near-consensus CAP site in vivo and found it to be diminished fourfold relative to wild type in each case. Activation of lac transcription by these mutant proteins remains the same as with wild-type CAP. Thus our results confirm that Glu171 in helix E of CAP is not involved directly in the activation of transcription. Yet CAP-E171K does not activate transcription as well as wild-type CAP under all circumstances. Possible reasons for this absence of activation are discussed.
Breul
A
A
Institut für Genetik, Universität zu Köln, FRG.
Assmann
H
H
Golz
R
R
von Wilcken-Bergmann
B
B
Müller-Hill
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005971:Glutamates; D018698:Glutamic Acid; D007786:Lactose Factors / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D002373:Cyclic AMP Receptor Protein; D005971:Glutamates; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase