Targeted protein footprinting: Where different transcription factors bind to RNA polymerase

Gene transcription is regulated through the interactions of RNA polymerase (RNAP) with transcription factors, such as the bacterial sigma proteins. We have devised a new strategy that relies on targeted protein footprinting t o make an extensive survey of proximity to the protein surface. This involv es attaching cutting reagents randomly to lysine residues on the surface of a protein such as sigma. The lysine-labeled sigma protein is then used to cleave the polypeptide backbones of the RNAP proteins at exposed residues a djacent to the sigma binding site. We used targeted protein footprinting to compare the areas near which sigma(70), sigma(54), sigma(38), sigma(E), Nu sA, GreA, and omega bind to the protein subunits of Escherichia coli RNAP. The sigma proteins and NusA cut sites in similar regions of the two large R NAP subunits, beta and beta', outlining a common surface. GreA cuts a large r set of sites, whereas omega shows no overlap with the others, cutting onl y the beta' subunit at a unique location.