| Gene transcription is regulated through the interactions of RNA polymerase (RNAP) with various transcription factors, such as the bacterial σ proteins. The RNAP holoenzyme of Escherichia coli is composed of the core enzyme and one of the multiple species of σ subunits. Each σ subunit is responsible for directing the holoenzyme to a different set of promoters, thereby acting in promoter selectivity and transcription initiation. The binding sites of regulatory proteins on core RNAP are not well understood, primarily due to a lack of experimental tools. We have developed an efficient method, targeted protein footprinting, for mapping regions near the binding site of one protein on another. This involves attaching cutting reagents randomly to surface accessible lysine residues on a protein, and then using this lysine-labeled protein to cleave the polypeptide backbone of the other protein at exposed residues adjacent to its binding site. We modify the native lysine side chains of a chosen protein in low yield with the cutting reagent iron (S)-1-( p-bromoacetanxidobenzyl) ethylenediaminetetraacetate (FeBABE) via a 2-iminothiolane (2IT) linker. Because lysines are common residues on the surfaces of most proteins, this new methodology makes it straightforward to label any number of proteins, allowing many different complexes to be studied in parallel.; This technique was first used to explore those regions of the σ 70 binding site on core RNAP that had not been accessed by cutting reagents attached to single-cysteine mutants. Targeted protein footprinting by lysine-labeled σ70 provided information equivalent to a large set of single-cysteine mutants. This has led us to explore and compare the interactions of several different proteins with RNAP. We used targeted protein footprinting to compare the areas near which σ 70, σ54, σ38, σ E, NusA, GreA and ω bind to the protein subunits of Escherichia coli RNAP. The σ proteins and NusA cut sites in similar regions of the two large RNAP subunits, β and β′ , outlining a common surface. GreA cuts a larger set of sites, whereas ω shows no overlap with the others, cutting only the β′ subunit at a unique location. |
