Conformational changes triggered by Mg²⁺ mediate transactivator function

Abstract

Transactivator protein C of bacteriophage mu is essential for the transition from middle to late gene expression during the phage life cycle. The unusual, multistep activation of mom promoter (P_mom) by C protein involves activator-mediated promoter unwinding to recruit RNA polymerase and subsequent enhanced promoter clearance of the enzyme. To achieve this, C binds its site overlapping the -35 region of the mom promoter with a very high affinity, in Mg²⁺-dependent fashion. Mg²⁺-mediated conformational transition in C is necessary for its DNA binding and transactivation. We have determined the residues in C which coordinate Mg²⁺, to induce allosteric transition in the protein, required for the specific interaction with DNA. Residues E26 and D40 in the putative metal binding motif (E₂₆X₁₀D₃₇X₂D₄₀₎ present toward the N-terminus of the protein are found to be important for Mg²⁺ ion binding. Mutations in these residues lead to altered Mg²⁺-induced conformation, compromised DNA binding, and reduced levels of transcription activation. Although Mg²⁺ is widely used in various DNA transaction reactions, this report provides the first insights on the importance of the metal ion-induced allosteric transitions in regulating transcription factor function.