Hadizadeh Nastaran, Johnson Reid C, Marko John F
Department of Physics and Astronomy, Northwestern University, Evanston, Illinois, USA.
Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
J Bacteriol. 2016 May 27;198(12):1735-42. doi: 10.1128/JB.00225-16. Print 2016 Jun 15.
Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or "facilitated dissociation." Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an "exchange" rate constant (kexch) of ≈10(4) M(-1) s(-1), comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo
Bacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolated E. coli chromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed in in vitro experiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function.
人们普遍认为，蛋白质从DNA双螺旋上的解离速率仅取决于最初结合的蛋白质-DNA复合物内部的相互作用，而不取决于附近分子的浓度。然而，最近的一些单DNA实验表明，解离速率取决于溶液中蛋白质的浓度，即“促进解离”。在这里，我们证明了这种效应在大肠杆菌主要类核蛋白Fis与分离的细菌染色体上发生。我们分离了大肠杆菌类核，并表明绿色荧光蛋白（GFP）-Fis的解离受溶液中Fis浓度的控制，其“交换”速率常数（kexch）约为10⁴ M⁻¹ s⁻¹，与单DNA实验中观察到的速率相当。我们还表明，这种效应强烈依赖于盐。我们的结果表明，在体内组装的染色体上体外可以观察到促进解离现象。
细菌是研究基因调控和染色体动力学的重要模型系统，这两者都从根本上依赖于蛋白质与DNA结合和解离的动力学。在对分离的大肠杆菌染色体进行的实验中，这项研究表明，多产的转录因子和染色体包装蛋白Fis从细菌染色体上的解离速率强烈依赖于Fis浓度，类似于在体外实验中观察到的情况。因此，游离的细胞DNA结合蛋白浓度可以强烈影响与染色体结合的蛋白质的寿命，在基因调控的定量考虑中必须予以考虑。这些结果对转录因子具有特别深远的意义，因为DNA结合寿命可能是调节功能的关键决定因素。
