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Nucleic Acids Research, 1984, Vol. 12, No. 20 7741-7752
© 1984

Articles

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

E. Bertrand-Burggraf, M. Schnarr, J.F. Lefevre and M. Daune

Laboratoire de Biophysique, Institut de Biologic Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084 Strasbourg Cédex, France

Received July 5, 1984. Accepted September 28, 1984.

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/rnole 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 topoisome rase I, we have determined the specific unwinding by RNA polymerase of the tet promoter of pBR322 (430°). This unwinding differs only slightly from the mean value (470°) obtained for all the promoters of pBR322.


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