Effect of superhelicity on the transcription from the tet promoter of pBR322. Abortive initiation and unwinding experiments

Abstract

Supercoiling of DNA is now known to have considerable effects on transcription in bacteria. By abortive initiation reaction (6) we have determined the binding constant KB and the forward rate of isomerization k2 as a function of temperature, pH and buffer for the tet promoter in a supercoiled plasmid. If the activation energy of isomerization is very similar to that obtained previously under the same conditions on a linearized plasmid (6) (respectively 21 +/- 5 kcal/mole and 13 +/- 5 kcal/mole) the supercoiling introduces very important and not well understood changes in the thermodynamic parameters of the association polymerase - promoter. Using the technique of superhelical DNA relaxation by eukaryotic topoisomerase I, we have determined the specific unwinding by RNA polymerase of the tet promoter of pBR322 (430 degrees). This unwinding differs only slightly from the mean value (470 degrees) obtained for all the promoters of pBR322.