A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K.
Understanding the role of Escherichia coli histone-like protein integration host factor (IHF) in replication of R6K plasmid (Dellis, S., and Filutowicz, M. (1991) J. Bacteriol. 173, 1279-1286) requires detailed analyses of the interaction of IHF protein with the plasmid's replication origin (gamma ori). We describe an electron microscopic analysis which shows that a compact structure can be formed in the presence of IHF, in which, on average, a 102-base pair (bp) ori segment is involved. IHF.gamma ori complexes also undergo a two-step conformational change in an IHF concentration-dependent manner when analysed by band shift assay. We believe that the DNA is bent at low IHF concentrations, but folded at high IHF concentrations. This idea is supported by the fact that electrophoretic mobility of the IHF.gamma ori complexes is faster at higher concentrations of IHF. Furthermore, it is shown that the formation of a compact nucleoprotein structure depends on the two regions flanking the AT-rich segment; the iterons to the right and the 106-bp ori domain to the left. Finally we show that IHF protects the entire AT-rich segment of the ori against nuclease cleavage. In addition to the protection, an altered cleavage pattern by DNase I, in the presence of high levels of IHF, was observed within the iterons but not within the 106-bp domain of the ori. Implications of the IHF-mediated gamma ori folding as a possible mechanism protecting the ori from replication inhibition by R6K initiator protein tau are discussed.
Filutowicz
M
M
Department of Bacteriology, University of Wisconsin, Madison 53706.
Inman
R
R
D001426:Bacterial Proteins / Q000378:metabolism* / Q000648:ultrastructure; D001483:Base Sequence; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D034982:Integration Host Factors; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D015183:Restriction Mapping; D009353:Zinostatin / Q000494:pharmacology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D009698:Nucleoproteins; D009838:Oligodeoxyribonucleotides; D009353:Zinostatin; D003850:Deoxyribonuclease I