Résumé
Formaldehyde is a highly reactive reagent which produces protein-DNA and protein-protein crosslinks b’tween macromolecules in close contact. TI1e distance between the’ crosslinking groups of protein and DNA should be around 2 A, suggesting an interaction at the range of van der Waals radii (1). Therefore, the formaldehyde crosslinking procedure provides a tool for identification of proteins and protein domains closely positioned to DNA. Formaldehyde crosslinking had been widely used for studies of protein-DNA interactions in chromatin in vivo and has been described in detail recently (2). In this chapter, the form is on applying formaldehyde crosslinking to studies of specific protein DNA interactions in vitro. This approach is principally different from the one mentioned above since relatively mild crosslinking conditions will be described that allow preferential formation of protein crosslinks of RNA polymerase from Escherichia coli with single-stranded (locally melted) regions of DNA in open complexes. In this context, crosslinking provides a simple way to identify domains on RNA polymerase bound to promoter ONA in open complexes as well as the identification of those subunit domains contacting the melted DNA. It can also be used for monitoring conformational changes in nucleoprotein complexes induced by different factors (temperature, salt conditions, pH) (3, 4). ln general, this approach can be applied to the study of any protein contacting dist011ed or melted regions of the DNA double helix.