NMR structure and functional studies of the Mu repressor DNA-binding domain

The repressor protein of bacteriophage Mu establishes and maintains lysogen y by shutting down transposition functions needed for phage DNA replication . It interacts with several repeated DNA sequences within the early operato r, preventing transcription from two divergent promoters. It also directly represses transposition by competing with the MuA transposase for an intern al activation sequence (IAS) that is coincident with the operator and requi red for efficient transposition. The transposase and repressor proteins com pete for the operator/IAS region using homologous DNA-binding domains locat ed at their amino termini. Here we present the solution structure of the am ino-terminal DNA-binding domain from the repressor protein determined by he teronuclear multidimensional nuclear magnetic resonance spectroscopy. The s tructure of the repressor DNA-binding domain provides insights into the mol ecular basis of several temperature sensitive mutations and, in combination with complementary experiments using flourescence anisotropy, surface plas mon resonance, and circular dichroism, defines the structural and biochemic al differences between the transposase and repressor DNA-binding modules. W e find that the repressor and enhancer domains possess similar three-dimens ional structures, thermostabilities, and intrinsic affinities for DNA, This latter result suggests that the higher affinity of the full-length repress or relative to that of the MuA transposase protein originates from cooperat ive interactions between repressor protomers and not from intrinsic differe nces in their DNA-binding domains. In addition, we present the results of n ucleotide and amino acid mutagenesis which delimits the minimal repressor D NA-binding module and coarsely defines the nucleotide dependence of repress or binding.