THE WING OF THE ENHANCER-BINDING DOMAIN OF MU-PHAGE TRANSPOSASE IS FLEXIBLE AND IS ESSENTIAL FOR EFFICIENT TRANSPOSITION

A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer, Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA, T his initial critical step is facilitated by the transient binding of t he N-terminal domain of MuA to an enhancer DNA element within the Mu g enome (called the internal activation sequence, IAS). Recently we solv ed the three-dimensional solution structure of the enhancer-binding do main of Mu phage transposase (residues 1-76, MUA(76)) and proposed a m odel for its interaction with the IAS element, Site-directed mutagenes is coupled with an in vitro transposition assay has been used to asses s the validity of the model, We have identified five residues on the s urface of MuA that are crucial for stable synaptic complex formation b ut dispensable for subsequent events in transposition. These mutations are located in the loop (wing) structure and recognition helix of the MUA(76) domain of the transposase and do not seriously perturb the st ructure of the domain, Furthermore, in order to understand the dynamic behavior of the MUA(76) domain prior to stable synaptic complex forma tion, we have measured heteronuclear N-15 relaxation rates for the unb ound MUA(76) domain, In the DNA free state the backbone atoms of the h elix-turn-helix motif are generally immobilized whereas the residues i n the wing are highly flexible on the pico- to nanosecond time scale, Together these studies define the surface of MuA required for enhancem ent of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally order ed only upon DNA binding.