THE ESCHERICHIA-COLI FIS PROTEIN PREVENTS INITIATION OF DNA-REPLICATION FROM ORIC IN-VITRO

Fis protein participates in the normal control of chromosomal replicat ion in Escherichia coil. However, the mechanism by which it executes i ts effect is largely unknown. We demonstrate an inhibitory Influence o f purified Fis protein on replication from oriC in vitro. Fis inhibits DNA synthesis equally well in replication systems either dependent up on or independent of RNA polymerase, even when the latter is stimulate d by the presence of HU or IHF. The extent of inhibition by Fis is mod ulated by the concentrations of DnaA protein and RNA polymerase; the m ore limiting the amounts of these, the more severe the inhibition by F is. Thus, the level of inhibition seems to depend on the ease with whi ch the open complex can be formed. Fis-mediated inhibition of DNA repl ication does not depend on a functional primary Fis binding site betwe en DnaA boxes R2 and R3 in oriC, as mutations that cause reduced bindi ng of Fis to this site do not affect the degree of inhibition. The dat a presented suggest that Fis prevents formation of an initiation-profi cient structure at oriC by forming an alternative, initiation-preventi ve complex. This indicates a negative role for Fis in the regulation o f replication initiation.