DNA-facilitated target search by nucleoproteins: Extension of a biosensor-surface plasmon resonance method.
DNA-facilitated target search by nucleoproteins: Extension of a biosensor-surface plasmon resonance method.
复制标题
DOI：
10.1016/j.ab.2021.114298
复制
发表时间：
2021-09-15
影响因子：
2.9
通讯作者：
Wilson WD
中科院分区：
文献类型：
--
作者：
Vo TD;Schneider AL;Poon GMK;Wilson WD
为了扩大生物传感器-SPR在核蛋白识别DNA方面的应用价值，我们报道了两个ETS家族转录因子ELK1和ETV6对DNA促进的靶标搜索的比较分析。ETS结构域是开发基于生物传感器的技术的一个有吸引力的系统，因为在高度保守的DNA结合基序中编码了广泛的物理化学性质。在生物传感器方法的基础上，蛋白质被定量隔离并以非特异性复合体的形式呈现给固定的同源DNA，我们评估了内在同源和非特异性亲和力对远程(节间)目标搜索的影响。DNA促进结合的平衡常数对蛋白质的固有结合特性很敏感，因此当通过转移与不转移非特异性DNA发生结合时，它们对同源DNA的相对特异性增强。结合和解离动力学的直接测量显示了激活的复合体的离子特征，这证明了DNA促进的解离，即使ELK1和ETV6只有一个DNA结合表面。在盐浓度掩盖了平衡时非特异性预结合的影响时，同源结合的解离动力学与不存在非特异性DNA的情况截然不同。这些结果进一步加强了在生理环境中DNA促进的远程易位的重要性。
To extend the value of biosensor-SPR in the characterization of DNA recognition by nucleoproteins, we report a comparative analysis of DNA-facilitated target search by two ETS-family transcription factors: Elk1 and ETV6. ETS domains represent an attractive system for developing biosensor-based techniques due to a broad range of physicochemical properties encoded within a highly conserved DNA-binding motif. Building on a biosensor approach in which the protein is quantitatively sequestered and presented to immobilized cognate DNA as nonspecific complexes, we assessed the impact of intrinsic cognate and nonspecific affinities on long-range (intersegmental) target search. The equilibrium constants of DNA-facilitated binding were sensitive to the intrinsic binding properties of the proteins such that their relative specificity for cognate DNA were reinforced when binding occurred by transfer vs. without nonspecific DNA. Direct measurement of association and dissociation kinetics revealed ionic features of the activated complex that evidenced DNA-facilitated dissociation, even though Elk1 and ETV6 harbor only a single DNA-binding surface. At salt concentrations that masked the effects of nonspecific pre-binding at equilibrium, the dissociation kinetics of cognate binding were nevertheless distinct from conditions under which nonspecific DNA was absent. These results further strengthen the significance of long-range DNA-facilitated translocation in the physiologic environment.
登录
查看更多内容
影响因子：
5.6
作者：
Pollak, Adam J.;Chin, Aaron T.;Reich, Norbert O.
通讯作者：
Reich, Norbert O.
影响因子：
14.9
作者：
Stephens DC;Kim HM;Kumar A;Farahat AA;Boykin DW;Poon GM
通讯作者：
Poon GM
影响因子：
5.6
作者：
Giuntoli, Rebecca D.;Linzer, Nora B.;Marko, John F.
通讯作者：
Marko, John F.
影响因子：
14.9
作者：
Stephens, Dominique C.;Poon, Gregory M. K.
通讯作者：
Poon, Gregory M. K.
影响因子：
4.2
作者：
Gilroy, Emma L.;Hicks, Matthew R.;Rodger, Alison
通讯作者：
Rodger, Alison