Abstract
The catabolite activator protein (CAP) binding sites of the Escherichia coli galactose and lactose operons were probed by hydroxyl radical footprinting. This method reveals each base that is protected by the bound protein. The patterns of protection seen for the primary CAP sites at gal and lac were virtually identical. In the presence of RNA polymerase the footprint of the second CAP molecule at gal was found to be very similar to those at the other two sites. This upstream site in gal align's perfectly with the lac CAP site with respect to the start of P1 transcription. Replacing most of the gal second CAP site DNA with heterologous sequences did not abolish binding although it became noticeably weaker. In vitro transcription studies of this hybrid gal promoter DNA further demonstrated the reduced affinity of the second CAP. These results are consistent with molecular models proposed for specific CAP binding and suggest that the second CAP at gal may be responsible for overall stimulation of transcription at this operon. Thus, in spite of differences in stoichiometry, the mechanisms of activation by CAP at gal and lac may be quite similar.
Highlights
The catabolite activator protein (CAP) binding sites of the Escherichia coli galactose and lactose operons were probedby hydroxyl radical footprinting
When DNA upstream of -60 is removed so that the second CAP cannot bind, a CAP molecule bound at the primary sitewill still induce RNA polymerase to bind atP1 and initiate trangal may beresponsible for overall stimulation of tran- scription
The catabolite activator protein (CAP)o’f Escherichia coli One common feature of these operons is the sequence of stimulates transcriptionfrom many promoters, notably those the CAP-binding sitew, hich is remarkably similarat all sites involved in sugarmetabolism
Summary
The catabolite activator protein (CAP) binding sites of the Escherichia coli galactose and lactose operons were probedby hydroxyl radical footprinting. In the presence of RNApolymerase the footprint of the second CAP molecule at gal was found to be very similar to those at the other two sites. These results are consistent with molecular modelsproposed for specific CAP binding and suggest that the second CAP at the lac operon controlregion spans the-50 t o -70 region [7]. When DNA upstream of -60 is removed so that the second CAP cannot bind, a CAP molecule bound at the primary sitewill still induce RNA polymerase to bind atP1 and initiate trangal may beresponsible for overall stimulation of tran- scription. That CAP may alter the isomerization rateas well [4]
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