Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016 India.
Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016 India.
Int J Biol Macromol. 2018 Feb;107(Pt B):2026-2033. doi: 10.1016/j.ijbiomac.2017.10.075. Epub 2017 Oct 14.
Cell division is compromised in DnaAcos mutant E. coli cells due to chromosome over-replication. In these cells, CedA acts as a regulatory protein and initiates cell division by a hitherto unknown mechanism. CedA, a double stranded DNA binding protein, interacts with various subunits of RNA polymerase complex, including rpoB. To reveal how this concert between CedA, rpoB and DNA brings about cell division in E. coli, we performed biophysical and in silico analysis and obtained mechanistic insights. Interaction between CedA and rpoB was shown by circular dichroism spectrometry and in silico docking experiments. Further, CedA and rpoB were allowed to interact individually to a selected DNA and their binding was monitored by fluorescence spectroscopy. The binding constants of these interactions as determined by BioLayer Interferometry clearly show that rpoB binds to DNA with higher affinity (K=<1.0E-12M) as compared to CedA (K=9.58E-09M). These findings were supported by docking analysis where 12 intermolecular H-bonds were formed in rpoB-DNA complex as compared to 4 in CedA-DNA complex. Based on our data we propose that in E. coli cells chromosome over-replication signals CedA to recruit rpoB to specific DNA site(s), which initiates transcription of cell division regulatory elements.
细胞分裂在 DnaAcos 突变型大肠杆菌细胞中受到损害，因为染色体过度复制。在这些细胞中，CedA 作为一种调节蛋白，通过一种迄今未知的机制启动细胞分裂。CedA 是一种双链 DNA 结合蛋白，与 RNA 聚合酶复合物的各种亚基相互作用，包括 rpoB。为了揭示 CedA、rpoB 和 DNA 之间的这种协同作用如何导致大肠杆菌中的细胞分裂，我们进行了生物物理和计算分析，并获得了机制见解。通过圆二色性光谱和计算对接实验显示了 CedA 与 rpoB 之间的相互作用。此外，允许 CedA 和 rpoB 分别与选定的 DNA 相互作用，并通过荧光光谱监测其结合。通过生物层干涉法确定的这些相互作用的结合常数清楚地表明，rpoB 与 DNA 的结合亲和力更高（K=<1.0E-12M），而 CedA 的结合亲和力较低（K=9.58E-09M）。这些发现得到了对接分析的支持，其中 rpoB-DNA 复合物中形成了 12 个分子间氢键，而 CedA-DNA 复合物中形成了 4 个氢键。基于我们的数据，我们提出在大肠杆菌细胞中，染色体过度复制会向 CedA 发出信号，使其招募 rpoB 到特定的 DNA 位点，从而启动细胞分裂调节元件的转录。
