THE FIS PROTEIN FAILS TO BLOCK THE BINDING OF DNAA PROTEIN TO ORIC, THE ESCHERICHIA-COLI CHROMOSOMAL ORIGIN

The Escherichia coil chromosomal origin contains several bindings site s for factor for inversion stimulation (FIS), a protein originally ide ntified to be required for DNA inversion by the Hin and Gin recombinas es, The primary FIS binding site is close to two central DnaA boxes th at are bound by DnaA protein to initiate chromosomal replication. Beca use of the close proximity of this FIS site to the two DnaA boxes, we performed in situ footprinting with 1,10-phenanthroline-copper of comp lexes formed with FIS and DnaA protein that were separated by native g el electrophoresis, These studies show that the binding of FIS to the primary FIS site did not block the binding of DnaA protein to DnaA box es R2 and R3, Also, FIS appeared to be bound more stably to oriC than DnaA protein, as deduced by its reduced rate of dissociation from a re striction fragment containing oriC, Under conditions in which FIS was stably bound to the primary FIS site, it did not inhibit oriC plasmid replication in reconstituted replication systems, Inhibition, observed only at high levels of FIS, was due to absorption by FIS binding of t he negative superhelicity of the oriC plasmid that is essential for th e initiation process.