Hydroxyl radical footprinting of DNA complexes of the ets domain of PU.1 and its comparison to the crystal structure.
Hydroxyl radical footprinting has been used to probe interactions in complexes between the ets domain of the murine transcription factor PU.1 and three different DNA restriction fragments, each containing one copy of the recognition sequence 5'-GGAA-3'. Two natural PU.1 binding sites, the SV40 enhancer site and the lambdaB motif of Ig lambda2-4 enhancer, were used as well as the PU.1 binding site present in the crystallized PU.1-DNA complex [Kodandapani, R., Pio, F., Ni, C.-Z., Piccialli, G., Klemsz, M., McKercher, S. R., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460]. The footprints obtained for the three different DNA sequences are almost identical. The extent of contact with the protein was monitored for every base in the complex. Two concentration-dependent cleavage sites on the complementary TTCC strand are evidence of a specific interaction between PU.1 and the DNA. Two more protection sites and a hypersensitive cleavage site on the GGAA strand were observed. Although these data confirm the global structure of the PU.1-DNA complex as suggested by crystallography, the footprinting data reveal differences between the protein-DNA contacts in solution and in the crystal state. An additional interaction site not present in the crystal structure was observed by hydroxyl radical footprinting.
Gross
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Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 2S2, Canada.
Arrowsmith
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Macgregor
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RB
D000818:Animals; D001665:Binding Sites; D016384:Consensus Sequence; D003461:Crystallography; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D017665:Hydroxyl Radical; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D000099232:Proto-Oncogene Protein Spi-1
D004247:DNA; D004268:DNA-Binding Proteins; D017665:Hydroxyl Radical; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1