Two alternative structures can be formed by IHF protein binding to the plasmid R6K gamma origin.
Escherichia coli integration host factor (IHF) contributes to the regulation of R6K plasmid copy number by counteracting the inhibitory activity of the plasmid-encoded replication protein pi. Two IHF-binding sites (ihf1 and ihf2) flank seven iterons in the origin which bind pi protein. As previously shown by electron microscopy, IHF can compact a large segment of the R6K gamma origin DNA, encompassing site ihf1, an AT-rich domain containing ihf1, and some of the seven iterons located downstream of ihf1. We termed this phenomenon IHF-mediated DNA folding. This folding requires a high IHF concentration, and the region of the origin (replication enhancer) located to the left of the AT-rich domain. However, site ihf2 is not necessary in forming the folded structure. As reported here, IHF binding to ihf2 can be detected in gel mobility shift assays only if the leftmost enhancer region is absent. Sites ihf1 and ihf2 each contain two consensus IHF sequences. Site-directed mutagenesis was performed to determine which sequences are recognized by IHF protein and which sites are involved in forming the various gamma origin-IHF complexes. Finally, we define the boundaries of protection from DNaseI digestion when IHF is bound to ihf2. We propose a model in which IHF protein bound to ihf1, in the absence of the enhancer region, facilitates IHF binding to ihf2.
Dellis
S
S
Department of Bacteriology, University of Wisconsin, Madison 53706.
Schatz
T
T
Rutlin
K
K
Inman
R B
RB
Filutowicz
M
M
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D034982:Integration Host Factors; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D011994:Recombinant Proteins; D003850:Deoxyribonuclease I