背景回顾：The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. 

提出问题：So far, ARF–ARF and ARF–DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. 

主要研究：Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). 

结果：We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. 

结论：Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

信号分子生长素主要通过调节基因的表达，以此来协调植物的多种生长和发育过程。其中，转录响应是由生长素响应因子家族（ARF）介导的。该家族的单体能够识别DNA基序，并可以通过其DNA结合域（DBD）进行同源二聚化，从而能够结合一对呈反向重复的结合位点。大多数ARF蛋白还包含一个C端PB1结构域，该结构域能够与抑制子Aux/IAA进行互作，从而介导ARF和Aux/IAA之间的相互作用。考虑到PB1结构域的双重作用，以及DBD和PB1两个结构域都能够介导二聚化的能力，随之而来的一个关键问题就是这些结构域如何作用于ARF蛋白的DNA结合特异性和亲和力。到目前为止，ARF–ARF以及ARF–DNA相互作用大多是使用定性方法来研究的，这些方法不能提供关于结合平衡的定量和动态信息。本文中，作者利用基于单分子荧光共振能量转移smFRET技术的DNA结合试验，研究了几个拟南芥ARF蛋白与一个IR7生长素响应元件（AuxRE）相互作用的亲和力和动力学。作者发现，AtARF2的DBD和PB1结构域都有助于DNA结合，并且作者鉴定到了ARF二聚体稳定性是决定AtARFs结合亲和力和动力学的关键参数。最后，作者推导了一个四态循环模型的解析解，该模型解释了AtARF2和IR7之间相互作用的动力学和亲和力。本文的研究结果表明，ARF对复合DNA响应元件的亲和力是由二聚化平衡决定的，并且这是影响ARF介导的转录活性的关键元件。

** Johannes Hohlbein **