Gross P, Arrowsmith C H, Macgregor R B
Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario M5S 2S2, Canada.
Biochemistry. 1998 Apr 14;37(15):5129-35. doi: 10.1021/bi972591k.
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.
羟基自由基足迹法已被用于探究小鼠转录因子PU.1的ets结构域与三个不同的DNA限制片段之间复合物中的相互作用，每个片段都含有一个5'-GGAA-3'识别序列拷贝。使用了两个天然的PU.1结合位点，即SV40增强子位点和Ig λ2-4增强子的λB基序，以及存在于结晶的PU.1-DNA复合物中的PU.1结合位点[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]。从三个不同DNA序列获得的足迹几乎相同。监测了复合物中每个碱基与蛋白质的接触程度。互补TTCC链上两个浓度依赖性切割位点证明了PU.1与DNA之间存在特异性相互作用。在GGAA链上还观察到另外两个保护位点和一个超敏切割位点。尽管这些数据证实了晶体学所提示的PU.1-DNA复合物的整体结构，但足迹数据揭示了溶液中和晶体状态下蛋白质-DNA接触之间的差异。通过羟基自由基足迹法观察到了晶体结构中不存在的一个额外相互作用位点。
