转录激活因子p53是一个肿瘤抑制蛋白，控制对细胞命运决策至关重要的细胞逮捕、细胞衰老和细胞凋亡等细胞通路。它通过与共调控复合物进行相互作用，与被称为响应元件（REs）的特定DNA序列结合，以控制转录活性。尽管其在生物学上的重要性，但p53结合REs的机制仍不清楚。为了解决这个问题，我们利用体外单分子荧光方法，在平衡条件下实时量化了全长人源p53与五个天然REs的动态结合。我们的方法还能够量化DNA结合的p53寡聚体状态。我们发现几乎没有证据表明在与p53四聚体/DNA复合物结合或解离的过程中，二聚体/DNA复合物会形成中间体。然而，有趣的是，在某些REs上，二聚体可以迅速从四聚体/DNA复合物中交换出来。实时动力学测量使我们能够确定所有五个REs中的结合和解离速率常数，从而揭示了两个在运动学上不同的四聚体p53/RE复合物种群。对于较不稳定的种群，解离的速率常数在最接近共识序列的REs上较大，表明更有利的结合序列形成运动学上最不稳定的复合物。总的来说，我们的单分子测量为四聚体p53在不同的天然REs上形成复合物的机制提供了新的见解。 版权所有：©2023 Suwita等。本文为开放获取文章，基于创作共用许可证，任何媒介下的使用、分发和复制都是允许的，只要提到原作者和来源。

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.Copyright: © 2023 Suwita et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.