E. coli RNA polymerase promoters on superhelical SV40 DNA are highly selective targets for chemical modification.

Abstract

Supercoiled DNAs are required or preferred in a number of biological processes such as repair and transcription. This requirement may be due to special properties which are present in supercoiled (FI) DNAs but not in the corresponding allomorphic forms. If sufficiently supercoiled, FI DNAs seem to contain single-stranded regions that are accessible to single-strand specific reagents and endonuclease1–3. The free energy associated with super helix formation may also be used to drive ligand–DNA or protein–DNA interactions3–7. In vitro studies have shown that superhelical DNA is a better template for transcription than its corresponding allomorphic forms8–15, and this is due probably to a change in the initial binding affinity of the RNA polymerase at promoter sites15–18. Previous work in our laboratory has suggested that the single-strand specific probe N-cyclohexyl-N′-β-(4-methylmorpholinium) ethyl carbodiimide (CMC), which almost totally inhibits transcription of SV40 and PM2 DNA14,18, is acting preferentially at promotor sites18–20. We report here experiments to determine the selectivity of CMC for promoters on superhelical SV40 DNA. After limited reaction with CMC there was substantially less Escherichia coli RNA polymerase bound to reacted SV40 DNA than to the unmodified DNA. Marked inhibition of transcription from the modified DNA demonstrated that CMC is highly selective for E. coli RNA polymerase promotors in superhelical DNA, suggesting that the introduction of superhelical turns may alter the parameters by which the enzyme interacts with DNA through promotor recognition and opening of the DNA.