Specific binding of CAP factor to lac promoter DNA

Abstract

CATABOLITE repression in Escherichia coli is mediated by the cyclic AMP dependent catabolite activator protein (CAP). Growth conditions where the induced catabolism of certain sugars is energetically favourable, primarily in the absence of glucose, promote the intracellular induction of a high cyclic AMP concentration; CAP binds cyclic AMP, is activated, and stimulates mRNA synthesis from promoters of catabolite repressible operons^1–4. The mechanism by which CAP influences the interaction between RNA polymerase and promoter DNA in unknown. Purified CAP preparations exhibit a cyclic AMP-dependent DNA binding activity, but efforts to detect specific binding to promoters of catabolite repressible genes have been unsuccessful; all DNAs tested have shown similar binding affinities^5,6. The only evidence for a specific interaction between CAP and promoter DNA is a fluorescent probe study which showed that a lac transducing phage DNA molecule induces a conformational change in CAP that is not seen with the wild-type phage molecule⁷; but no effort was made to correlate this difference with in vivo CAP behaviour. Here I report experiments which demonstrate that the CAP factor binds specifically to a site within the lac promoter. This binding is sensitive to mutations which affect the ability of CAP to stimulate in vivo and in vitro lac gene expression.