Carbodiimide inactivation of Escherichia coli RNA polymerase promoters on supercoiled simian virus 40 and ColE1 DNAs occurs by a one-hit process at salt concentrations in the physiological range

Abstract

A previous study (Hale, P., Woodward, R. W., and Lebowitz, J. (1980) Nature 284, 640-644) showed that Escherichia coli RNA polymerase promoters on superhelical SV40 DNA are highly selective targets for chemical modification by the water-soluble carbodiimide, N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethyl carbodiimide (CMC). To extend the inactivation analysis of supercoiled DNAs, we determined the number and location of RNA polymerase binding sites on the supercoiled and linear forms of ColE1 DNA. We also determined the site distribution of [3H] CMC on the superhelical form. This information, coupled with per cent inhibition of transcription versus CMC-bound curves, allowed a test of the specificity of the CMC inactivation by the Poisson equation. Curves were obtained for supercoiled SV40 DNA modified at 0 and 100 mM NaCl (2 mM NaPi, pH 7.0) and for supercoiled ColE1 DNA modified at 0, 100, and 320 mM NaCl. For supercoiled SV40 DNA, these data, coupled to our knowledge of the number of RNA polymerase binding sites from the study cited above, revealed an excellent fit to a one-hit inactivation by the Poisson equation for DNA modified at 100 mM NaCl. For ColE1 DNA, we obtained an excellent fit to a Poisson distribution when supercoiled DNA was modified at 320 mM NaCl. The Poisson distribution can be applied to [3H] CMC restriction fragment data with equivalent results. These results suggest that promoter sites can be forced into different structural conformations with variable degrees of unpairing.