Zhou Daniel H, Jeon Jongcheol, Farheen Nida, Friedman Larry J, Kondev Jane, Buratowski Stephen, Gelles Jeff
Department of Biochemistry, Brandeis University, Waltham, MA 02453.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
bioRxiv. 2024 Dec 11:2024.12.10.627625. doi: 10.1101/2024.12.10.627625.
Transcription activators trigger transcript production by RNA Polymerase II (RNApII) via the Mediator coactivator complex. Here the dynamics of activator, Mediator, and RNApII binding at promoter DNA were analyzed using multi-wavelength single-molecule microscopy of fluorescently labeled proteins in budding yeast nuclear extract. Binding of Mediator and RNApII to the template required activator and an upstream activator sequence (UAS), but not a core promoter. While Mediator and RNApII sometimes bind as a pre-formed complex, more commonly Mediator binds first and subsequently recruits RNApII to form a preinitiation complex precursor (pre-PIC) tethered to activators on the UAS. Interestingly, Mediator occupancy has a highly non-linear response to activator concentration, and fluorescence intensity measurements show Mediator preferentially associates with templates having at least two activators bound. Statistical mechanical modeling suggests this "synergy" is not due to cooperative binding between activators, but instead occurs when multiple DNA-bound activator molecules simultaneously interact with a single Mediator.
转录激活因子通过中介辅激活复合物触发RNA聚合酶II（RNApII）的转录产物生成。在此，利用芽殖酵母核提取物中荧光标记蛋白的多波长单分子显微镜技术，分析了激活因子、中介物和RNApII在启动子DNA上的结合动力学。中介物和RNApII与模板的结合需要激活因子和上游激活序列（UAS），但不需要核心启动子。虽然中介物和RNApII有时会以预先形成的复合物形式结合，但更常见的情况是中介物先结合，随后招募RNApII形成与UAS上的激活因子相连的预起始复合物前体（pre-PIC）。有趣的是，中介物占据率对激活因子浓度具有高度非线性响应，荧光强度测量表明中介物优先与至少结合有两个激活因子的模板结合。统计力学模型表明，这种“协同作用”并非由于激活因子之间的协同结合，而是当多个与DNA结合的激活因子分子同时与单个中介物相互作用时发生的。
