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Nucleic Acids Research, 1991, Vol. 19, No. 4 833-840
© 1991

MOLECULAR BIOLOGY

Protein-DNA cross-linking at the lac promoter

Malcolm Buckle, Johannes Geiselmann, Annie Kolb and Henri Buc*

Institut Pasteur Unité de Physicochimie des Macromolécules Biologiques (URA 1149 du CNRS), F-75724 Paris CEDEX 15. France

* To whom correspondence should be addressed

Received October 22, 1990. Revised January 28, 1991. Accepted January 28, 1991.

We report the results of photo-cross-linking of RNA polymerase and the cyclic AMP receptor protein (CRP) to the lac UV5 promoter region carried on either a linear fragment or a supercoiled plasmid. We have devised a protocol that allows the localisation of bases in contact with the protein. RNA polymerase makes contacts within the –10 and –35 regions of the promoter, essentially on the non-template strand. The CRP contact points found in a binary complex are affected by the formation of the ternary complex containing RNA polymerase. Supetcoiling has no effect on the position of contacts in any of the complexes. These conclusions were derived from experiments performed using a generally applicable, non-interfering technique that reveals direct contacts between proteins and nucleic acids in nucleoprotein complexes.


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