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 function, is regulated in a complex manner at both transcriptional and translational levels. The phage-encoded C protein functions as an activator of mom transcription. The mom promoter has features of an activator-dependent weak promoter, and the C binding site is located upstream and overlapping the -35 region and includes the palindromic sequence TTAT(N)6ATAA. The interactions of this activator protein at its binding site in Pmom 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. Dimethylsulfate 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 observed with hydroxyl radical, generated by Fe(II)-EDTA, which shows the binding of the protein to one face of the helix. The extent of protection conferred by the bound protein, however, is not uniform, suggesting that the interaction is asymmetric. The chemical nuclease 1,10-phenanthroline-copper, a minor groove specific ligand, shows hyper-reactivity upon protein binding in the top strand nucleotide triplet CAC, again confirming the protein-induced alterations in DNA conformation. Gel exclusion 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 electrophoretic mobility shift assay using heterodimer of pure C protein and staphylococcal protein A-C fusion. The deletion analysis implicates a role for the carboxyl-terminal region of the protein in DNA binding.
Ramesh
V
V
Centre for Genetic Engineering, Indian Institute of Science, Bangalore, India.
Nagaraja
V
V
D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002850:Chromatography, Gel / Q000379:methods; D003432:Cross-Linking Reagents; D018983:DNA Footprinting / Q000379:methods*; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005374:Filtration; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017384:Sequence Deletion; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C090591:C protein, Bacteriophage Mu; D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014764:Viral Proteins