Physico‐chemical Properties of a DNA Binding Protein: Escherichia coli Factor H1

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Abstract
H1 factor is a heat-stable protein found in large amounts in E. coli. In vitro, this protein stimulated transcription of [phage] .lambda. templates by E. coli DNA-dependent RNA polymerase [EC 2.7.7.6]. The subunit MW of this factor was re-estimated and was 15,500 .+-. 1000 in the presence of 2-mercaptoethanol. Crosslinking experiments performed with dimethylsuberimidate in the presence or absence of the DNA template indicate the presence of multiples of the 15,500 MW subunit up to the tetramer, the dimeric species being predominant. One cysteinyl residue/15,000 MW subunit is labeled by 4-chloro-7-nitrobenzofurazan. This residue is not labeled if the factor is exposed to oxidizing conditions. In this case, 3 lysyl residues are titrated. H1 factor behaves as a DNA-binding protein. Binding to DNA was detected by 2 independent methods: displacement of a fluorescently labeled factor by the native protein and retention of radioactive DNA on millipore filters in the presence of the factor. Under experimental conditions (high ionic strength, absence of Mg2+), the saturation function of .lambda. plac DNA and of wild-type .lambda. DNA was non-cooperative. Saturation is reached when 300 .+-. 30 molecules of dimeric factor are bound/.lambda. molecule, the average Kd of the complex being 10 nM. The dissociation time of the H1.cntdot.DNA complex is < 5 s at 37.degree. C. The binding of this factor lowers the affinity of native DNA for ethidium bromide. In the presence of this intercalating dye, the solubility of the complex decreases drastically.