Footprinting DNA-protein complexes in situ following gel retardation assays using 1,10-phenanthroline-copper ion: Escherichia coli RNA polymerase-lac promoter complexes.
Protein-DNA complexes isolated in gel retardation assays can be digested within the acrylamide matrix by the nuclease activity of 1,10-phenanthroline-copper ion (OP-Cu). When the oligonucleotide products are eluted and analyzed on a sequencing gel, a footprint of the DNA-protein complex is obtained. Therefore, any protein-DNA complex isolated by the widely used gel retardation technique can be defined in terms of sequence-specific interactions by this simple methodology. The binding of the lac repressor and Escherichia coli RNA polymerase to an EcoRI fragment containing the lac control region has been studied by the combined gel retardation-1,10-phenanthroline-copper ion footprinting procedure. Footprints of lac repressor binding correspond to those obtained in solution with OP-Cu and DNase I and verify the experimental procedures. In studying E. coli RNA polymerase-promoter complexes, we have found that magnesium ion is required to form single-stranded DNA structures characteristic of kinetically competent open transcription complexes.
Kuwabara
M D
MD
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024-1570.
Sigman
D S
DS
D002850:Chromatography, Gel; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D010618:Phenanthrolines; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism
D010618:Phenanthrolines; D012097:Repressor Proteins; C038556:bis(1,10-phenanthroline)copper(1+) ion; C014741:bis(1,10-phenanthroline)copper(2+) ion; D012321:DNA-Directed RNA Polymerases