title: The sequence-specific association of the ETS domain of murine PU.1 with DNA exhibits unusual energetics.
abstract: PU.1 belongs to the ETS family of transcription factors whose DNA-binding domains recognize purine-rich sequences containing the consensus 5'-GGAA/T-3'. We have characterized the sequence-specific association of the ETS domain of murine PU.1 to the lambdaB site of the Ig(lambda)2-4 enhancer as a function of temperature and pH by electrophoretic mobility shift, filter binding, and CD spectroscopy. From 0 to 25 degrees C, the dissociation equilibrium constant KD is, within experimental uncertainty, insensitive to temperature, and is only a weak function of temperature from 25 to 52 degrees C. van't Hoff analysis yielded a small value of DeltaCp = -2.1 kJ x mol(-1) x K(-1) in phosphate buffer, pH 7.4, containing 250 mM Na+. KD also shows a weak dependence at 25 degrees C on pH from 6.7 to 9.0 in phosphate, cacodylate, and Tris buffers that have disparate heats of ionization. The CD spectrum of the protein-DNA complex could be accounted for by a simple linear combination of the spectra of the free components throughout the binding temperature range. Structural calculations indicate that dehydration of solvent-accessible contact surfaces on the protein and DNA accounts for up to DeltaCp approximately -1 kJ x mol(-1) x K(-1). Taken together, these observations suggest that the hydrophobic effect and, in particular, coupled folding do not contribute significantly to the energetics of sequence-specific association. This is unusual with respect to other sequence-specific protein-DNA interactions for which significant enthalpic contributions and large negative heat capacities are commonly observed.
authors.affiliation: Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 2S2, Canada.
authors.forename: Gregory M K
authors.initials: GM
authors.lastname: Poon
authors.forename: Petra
authors.initials: P
authors.lastname: Gross
authors.forename: Robert B
authors.initials: RB
authors.lastname: Macgregor
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007146:Immunoglobulin lambda-Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D004268:DNA-Binding Proteins; D007146:Immunoglobulin lambda-Chains; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1