Repression and catabolite gene activation in the araBAD operon.

AUTOR(ES)

Lichenstein, H S

RESUMO

Catabolite gene activation of the araBAD operon was examined by using catabolite gene activator protein (CAP) site deletion mutants. A high-affinity CAP-binding site between the divergently orientated araBAD and araC operons has been previously identified by DNase I footprinting techniques. Subsequent experiments disagreed as to whether this site is directly involved in stimulating araBAD expression. In this paper, we present data showing that deletions generated by in vitro mutagenesis of the CAP site led to a five- to sixfold reduction in single-copy araBAD promoter activity in vivo. We concluded that catabolite gene activation of araBAD involves this CAP site. The hypothesis that CAP stimulates the araBAD promoter primarily by relieving repression was then tested. The upstream operator araO2 was required for repression, but we observed that the magnitude of CAP stimulation was unaffected by the presence or absence of araO2. We concluded that CAP plays no role in relieving repression. Other experiments showed that when CAP binds it induces a bend in the ara DNA; similar bending has been reported upon CAP binding to lac DNA. This conformational change in the DNA may be essential to the mechanism of CAP activation.

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