Determining the Architecture of a Protein-DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures,...

Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex.