SEQUENCE-SPECIFIC DNA-BINDING OF THE PHAGE-MU C-PROTEIN - FOOTPRINTING ANALYSIS REVEALS ALTERED DNA CONFORMATION UPON PROTEIN-BINDING

The mom gene of bacteriophage Mu, which codes for a DNA modification f unction, is regulated in a complex manner at both transcriptional and translational levels. The phage-encoded C protein functions as an acti vator of mom transcription. The mom promoter has features of an activa tor-dependent weak promoter, and the C binding site is located upstrea m and overlapping the -35 region and includes the palindromic sequence TTAT(N)(6)ATAA. The interactions of this activator protein at its bin ding site in P-mom has been investigated using four different chemical footprinting reagents. The protein footprint spans a region of 18 to 25 bp, depending on the nature of the chemical reagent used. Dimethyls ulfate protection experiments revealed the base-specific interactions. The protected guanines are separated by 15 bp and are located beyond the interrupted palindromic sequence. A tripartite footprint was obser ved with hydroxyl radical, generated by Fe(II)-EDTA, which shows the b inding of the protein to one face of the helix. The extent of protecti on conferred by the bound protein, however, is not uniform, suggesting that the interaction is asymmetric. The chemical nuclease 1,10-phenan throline-copper, a minor groove specific ligand, shows hyper-reactivit y upon protein binding in the top strand nucleotide triplet CAC, again confirming the protein-induced alterations in DNA conformation, Gel e xclusion chromatography and chemical crosslinking experiment with the purified protein suggest that this mode of interaction is accomplished by a dimeric protein. This observation is supported by electrophoreti c mobility shift assay using heterodimer of pure C protein and staphyl ococcal protein A-C fusion. The deletion analysis implicates a role fo r the carboxyl-terminal region of the protein in DNA binding. (C) 1996 Academic Press Limited