Physico-chemical properties of a DNA binding protein: Escherichia coli factor H1.
H1 factor is a heat-stable protein found in large amounts in Escherichia coli. In vitro, this protein has been found to stimulate transcription of lambda templates by E. coli DNA-dependent RNA polymerase. The subunit molecular weight of this factor has been re-estimated and found to be 15500 +/- 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 15500-Mr subunit up to the tetramer, the dimeric species being predominant. One cysteinyl residue per 15000-Mr subunit is labeled by 4-chloro-7-nitrobenzofurazan. This residue is not labeled if the factor is exposed to oxidizing conditions. In this case, three lysyl residues are titrated. H1 factor behaves as a DNA-binding protein. We have detected binding to DNA by two 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 our experimental conditions (high ionic strength, absence of magnesium ions), the saturation function of lambda plac DNA as well as of wild-type lambda DNA has been found to be non-cooperative. Saturation is reached when 300 +/- 30 molecules of dimeric factor are bound per lambda molecule, the average dissociation constant of the complex being 10nM. The dissociation time of the H1.DNA complex is less than 5 s at 37 degrees 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.
D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003852:Deoxyribonucleoproteins; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008970:Molecular Weight; D013053:Spectrophotometry; D013698:Templates, Genetic; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004279:DNA, Viral; D003852:Deoxyribonucleoproteins; D046911:Macromolecular Substances; D012321:DNA-Directed RNA Polymerases