The experiments presented in this proposal are designed to provide a better understanding of the biochemical basis of termination of DNa replication in Escherichia coli. Two components of the E. coli termination system have been identified: a DNA component, which consists of a 23 basepair sequence known as the terminator sequence, and a protein component called Tus, which binds to the DNA terminator sequence. It is proposed to investigate the protein-DNA interactions between Tus and the replisome. Specifically, it is proposed to: 1. Purify and characterize the Tus protein using column chromotography techniques. 2. Study the binding interactions between wild-type Tus protein and the chromosomal terminator sequences using a filter-binding assay. 3. Systematically analyze the sequence requirements for binding of Tus to the terminator sequences by generating site-specific mutations and determining the effect these mutations have on binding. A correlation between binding affinity and termination activity will also be established. 4. Generate mutant Tus proteins to define the active sites for DNA binding and termination. 5. Study the mechanism of replication inhibition using genetic and biochemical methods.
