Poon Gregory M K, Macgregor Robert B
Department of Pharmaceutical Sciences, University of Toronto, 19 Russell Street, M5S 2S2, Toronto, Ont., Canada.
J Mol Biol. 2004 Jan 2;335(1):113-27. doi: 10.1016/j.jmb.2003.09.046.
The ETS domain of the transcription factor PU.1 tolerates a large number of DNA cognate variants that differ exclusively in the sequences flanking a critical central consensus, 5'-GGAA-3'. We investigated the thermodynamics of site selection by the DNA-binding domain by following the PU.1 ETS/DNA equilibrium with a large set of cognate variants under various temperature and salt conditions by filter binding. Our results indicate that the stability of the ETS/DNA complex is quantitatively tied to variations in the change in heat capacity. Thermodynamic effects induced by changing Na(+) concentrations from 150 mM to 250 mM are complex and not readily interpreted by polyelectrolyte theory. We also extended our understanding of data from our previous investigation on energetic base-neighbour coupling, by dissecting the thermodynamic contributions underlying the observed free-energy coupling. In conjunction with available structural and biochemical data, we propose that site selectivity by the PU.1 ETS domain arises from differential protein/DNA contacts in the flanking sequences that modulate the orientation of the ETS recognition helix and trigger a coupled reduction in the flexibility observed in the unbound ETS domain.
转录因子PU.1的ETS结构域能够耐受大量仅在关键核心共有序列5'-GGAA-3'侧翼序列上存在差异的DNA同源变体。我们通过在各种温度和盐条件下利用一大组同源变体通过滤膜结合追踪PU.1 ETS/DNA平衡，研究了DNA结合结构域的位点选择热力学。我们的结果表明，ETS/DNA复合物的稳定性在数量上与热容变化相关。将Na(+)浓度从150 mM变为250 mM所诱导的热力学效应是复杂的，并且不易用聚电解质理论来解释。我们还通过剖析观察到的自由能耦合背后的热力学贡献，扩展了我们对先前关于能量碱基邻位耦合研究数据的理解。结合现有的结构和生化数据，我们提出PU.1 ETS结构域的位点选择性源于侧翼序列中不同的蛋白质/DNA接触，这些接触调节了ETS识别螺旋的方向，并引发了未结合的ETS结构域中观察到的灵活性的耦合降低。
