Protein-DNA cross-linking at the lac promoter.
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. Supercoiling 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.
Buckle
M
M
Institut Pasteur, Unité de Physicochimie des Macromolécules Biologiques (URA 1149 du CNRS), Paris, France.
Geiselmann
J
J
Kolb
A
A
Buc
H
H
D001483:Base Sequence; D003432:Cross-Linking Reagents; D004247:DNA / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases