Conformational dynamics of a transposition repressor in modulating DNA binding

The repressor of bacteriophage Mu functions in the establishment and mainte nance of lysogeny by binding to Mu operator DNA to shut down transposition. A domain at its N terminus functions in DNA binding, and temperature-sensi tive mutations in this domain can be suppressed by truncations at the C ter minus. To understand the role of the C-terminal tail in DNA binding, a fluo rescent probe was attached to the C terminus to examine its environment and its movement with respect to the DNA binding domain. The emission spectrum of this probe indicated that the C terminus was in a relatively hydrophobi c environment, comparable to the environment of the probe attached within t he DNA-binding domain. Fluorescence of two tryptophan residues located with in the DNA-binding domain was quenched by the probe attached to the C termi nus, indicating that the C terminus is in close proximity to this domain. A ddition of DNA, even when it did not contain operator DNA, reduced quenchin g of tryptophan fluorescence, indicating that the tail moves away from the DNA-binding domain as it interacts with DNA. The presence of the tail also produced a trypsin hypersensitive site within the DNA-binding domain; mutan t repressors with an altered or truncated C terminus were relatively resist ant to cleavage at this site. Interaction of the wild-type repressor with D NA greatly reduced cleavage at the site. A repressor with a temperature-sen sitive mutation in the DNA-binding domain was especially sensitive to cleav age by trypsin even in the presence of DNA, and the C-terminal tail failed to move in the presence of DNA at elevated temperatures. These results indi cate that the tail sterically inhibits DNA binding and that it moves during establishment of repression. Such conformational changes are likely to be involved in communication between repressor protomers for cooperative DNA b inding. (C), 2001 Academic Press.