CRP-DNA COMPLEXES - INDUCING THE A-LIKE FORM IN THE BINDING-SITES WITH AN EXTENDED CENTRAL SPACER

The consensus DNA sequence for binding of the Escherichia coli cyclic AMP receptor protein (CRP) has two symmetrically related inverted reco gnition elements TGTGA:TCACA, separated by a variable spacer, normally 6 bp long. We have shown that the CRP-cAMP complex, when bound to syn thetic binding sites with an extended 8 bp spacer segment, induces an increase in the DNA circular dichroism (CD). The CD change at lambda > 275 nm agrees with the shift of approximately one helical turn of DNA into A-like form. The B-conformation is preserved for CRP binding sit es similar to that in the Inc and uxaCA promoters with 6 bp spacers. A nother effect accompanying DNA binding is a dramatic increase of the n egative CD magnitude in the spectral region of the ligand cAMP, at lam bda < 272 nm. This effect is observed when CRP binds to specific sites with 6 or 8 bp spacers as well as to non-specific DNA. We reason that the A-like form arises by compressing and unwinding the DNA in CRP-DN A complexes having 8 bp central spacers. This serves to maintain a fix ed length and twisting angle anal is controlled by the protein's relat ively rigid frame. This model is consistent with the observation that some binding sites with 6 bp spacers may also show the CD increase inh erent to the sites with the extended 8 bp spacers. These 6 bp spacers are characterized by an increased twisting angle that requires their u nwinding to bind to CRP. We propose that a mutual adaptation between C RP and binding sites by local untwisting and a B-->A-like transition i n the DNA is of general importance and may occur in other protein-DNA complexes, such as the complex of RNA polymerase with promoter DNA.