Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements.
Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements.
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单分子研究表征了四聚体p53与不同天然反应元件结合的动力学机制。
DOI：
10.1371/journal.pone.0286193
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发表时间：
2023
期刊：
影响因子：
3.7
通讯作者：
Kugel, Jennifer F. F.
中科院分区：
文献类型：
--
作者：
Suwita, Johannes P. P.;Voong, Calvin K. K.;Ly, Elina;Goodrich, James A. A.;Kugel, Jennifer F. F.
转录激活蛋白P53是一种肿瘤抑制蛋白，它控制着对细胞命运决定至关重要的细胞通路，包括细胞周期停滞、衰老和凋亡。它作为四聚体与被称为反应元件(RES)的特定DNA序列结合，通过与共调节复合体的相互作用来控制转录。尽管P53具有重要的生物学意义，但其结合Res的机制仍不清楚。为了解决这个问题，我们使用了体外单分子荧光方法来实时定量在平衡条件下全长的人P53与五个天然RE的动态结合。我们的方法使我们能够量化DNA结合的p53的寡聚状态。我们发现几乎没有证据表明二聚体/DNA复合体在与P53四聚体/DNA复合体结合或解离的过程中作为中间体形成。然而，有趣的是，在某些分辨率下，二聚体可以迅速从四聚体/DNA复合体中交换出来。实时动力学测量使我们能够确定所有五个RE的结合和解离的速率常数，这揭示了两个动力学上不同的四聚体P53/RE复合体。对于不稳定的群体，解离速率常数在最接近共识的Res处较大，表明越有利的结合序列形成的动力学稳定的络合物越不稳定。总之，我们的单分子测量为四聚体P53在不同的天然RE上形成复合体的机制提供了新的见解。
The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Despite its biological importance, the mechanism by which p53 binds REs remains unclear. To address this, we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of full-length human p53 to five native REs in real time under equilibrium conditions. Our approach enabled us to quantify the oligomeric state of DNA-bound p53. We found little evidence that dimer/DNA complexes form as intermediates en route to binding or dissociation of p53 tetramer/DNA complexes. Interestingly, however, at some REs dimers can rapidly exchange from tetramer/DNA complexes. Real time kinetic measurements enabled us to determine rate constants for association and dissociation at all five REs, which revealed two kinetically distinct populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing that the more favorable binding sequences form the least kinetically stable complexes. Together our single molecule measurements provide new insight into mechanisms by which tetrameric p53 forms complexes on different native REs.
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