CAP and RNA polymerase interactions with the lac promoter: binding stoichiometry and long range effects.

The binding stoichiometries of the complexes formed when the E. coli cyclic AMP receptor protein (CAP) binds to 203 bp lac promoter-operator restriction fragments have been determined. Under quantitative binding conditions, a single dimer of CAP occupies each of two sites in the promoter. Different electrophoretic mobilities are observed for 1:1 complexes formed with L8-UV5 mutant, L305 mutant, and wild type promoter fragments, indicating sequence-specific structural differences between the complexes. The differences in gel mobility between L8-UV5 and wild type complexes disappear when the promoter fragments are cleaved with Hpa II restriction endonuclease. Models in which CAP alters DNA conformation or in which CAP forms a transient intramolecular bridge between two domains of a DNA molecule could account for these observations. The selective binding of RNA polymerase to CAP-promoter complexes is demonstrated: the binding of a single CAP dimer to the promoter is sufficient to stimulate subsequent polymerase binding. Functional CAP molecules are not released from the promoter on polymerase binding.     

The interaction of RNA polymerase and lac repressor with the lac control region.

We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex.     

The catabolite activator protein stabilizes its binding site in the E. coli lactose promoter.

The effect of catabolite activator protein, CAP, on the thermal stability of DNA was examined. Site specific binding was studied with a 62 bp DNA restriction fragment containing the primary CAP site of the E. coli lactose (lac) promoter. A 144 bp DNA containing the lac promoter region and a 234 bp DNA from the pBR322 plasmid provided other DNA sites. Thermal denaturation of protein-DNA complexes was carried out in a low ionic strength solvent with 40% dimethyl sulfoxide, DMSO. In this solvent free DNA denatured below the denaturation temperature of CAP. The temperature stability of CAP for site specific binding was monitored using an acrylamide gel electrophoresis assay. Results show that both specific and non-specific CAP binding stabilize duplex DNA. Site specific binding to the 62 bp DNA produced a 13.3 degrees C increase in the transition under conditions where non-specific binding stabilized this DNA by 2-3 degrees C.