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Protein-DNA cross-linking at the lac promoter
M Buckle1, J Geiselmann, A Kolb
1Institut Pasteur, Unité de Physicochimie des Macromolécules Biologiques (URA 1149 du CNRS), Paris, France.
Nucleic Acids Research
|February 25, 1991
Summary
This study reveals direct contacts between RNA polymerase and cyclic AMP receptor protein (CRP) on the lac UV5 promoter using a novel photo-cross-linking technique. Protein interactions and DNA supercoiling influence these specific DNA-protein binding sites.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Understanding DNA-protein interactions is crucial for gene regulation.
- The lac UV5 promoter is a well-studied model system for transcription initiation.
- RNA polymerase and CRP are key regulators of gene expression.
Purpose of the Study:
- To map precise DNA contact sites for RNA polymerase and CRP on the lac UV5 promoter.
- To investigate the influence of complex formation (binary vs. ternary) and DNA topology on these contacts.
- To introduce a versatile method for analyzing protein-DNA interactions in nucleoprotein complexes.
Main Methods:
- Photo-cross-linking of proteins to DNA.
- Localization of protein-DNA contact points on linear and supercoiled DNA fragments.
- Analysis of protein binding in binary and ternary complexes.
Main Results:
- RNA polymerase contacts were localized to the -10 and -35 regions, primarily on the non-template strand.
- Cyclic AMP receptor protein (CRP) contact sites shifted upon formation of the ternary complex with RNA polymerase.
- DNA supercoiling did not alter the observed protein-DNA contact positions.
Conclusions:
- The study provides a detailed map of protein-DNA interactions at the lac UV5 promoter.
- The findings highlight the dynamic nature of CRP binding in response to RNA polymerase presence.
- The developed technique offers a powerful tool for studying protein-nucleic acid interactions in various biological contexts.