Transcription Factor Mobility

用荧光漂白后恢复法（FRAP）研究了培养的腺癌3617细胞核内GFP标记的糖皮质激素受体（GFP-GR）的迁移率。GFP-GR是一种蛋白质转录因子，是细胞核DNA内小鼠乳腺肿瘤病毒（MMTV）启动子位点的配体。为了定量解释FRAP测量结果，开发了数学模型来描述GFP-GR的核内结合和扩散对荧光恢复的影响。这些模型包括低亲和力非特异性结合到分布在整个细胞核的位点，以及高亲和力结合到离散阵列的启动子位点。该模型考虑到核的有限体积。有限元软件被用来获得在两个空间维度的控制方程的数值解，以适应高亲和力网站的空间定位。通过与一维解析解的模拟结果进行比较，验证了二维数值解的有效性。参数敏感性分析表明，忽略扩散会导致约束参数估计的严重误差。该模型被应用于FRAP测量GFP-GR的高亲和力结合区域代表了一个本地化的重复MMTV启动子靶位点阵列。一个结果是对转录因子在启动子位点的停留时间上限的亚秒估计，这是令人惊讶的，因为转录可以持续数小时的时间尺度。这一发现提出了关于快速交换在转录机制和调控中的作用的问题。这表明，正在开发的FRAP技术将导致这些复杂的细胞内过程的更好的理解。

The mobility of GFP-tagged glucocorticoid receptor (GFP-GR) within the nucleus of cultured adenocarcinoma 3617 cells is being studied by fluorescence recovery after photobleaching (FRAP). GFP-GR is a protein transcription factor that is a ligand for mouse mammary tumor virus (MMTV) promoter sites within the nucleus DNA. To permit quantitative interpretation of the FRAP measurements, mathematical models were developed to describe the influence of intranucleus binding and diffusion of GFP-GR on the fluorescence recovery. The models include low-affinity non-specific binding to sites that are distributed throughout the nucleus, as well as high-affinity binding to a discrete array of promoter sites. The models take into account the finite volume of the nucleus. Finite element software is used to obtain numerical solutions of the governing equations in two spatial dimensions to accommodate the spatial localization of the high-affinity sites. The two-dimensional numerical solutions are validated by comparison to simulations from one-dimensional analytical solutions. Parameter sensitivity analysis indicates that ignoring diffusion can produce serious errors in binding parameter estimation. The models are being applied to FRAP measurements of GFP-GR with the high affinity binding region representing a localized array of repeated MMTV promoter target sites. One result was a sub-second estimate for the upper bound on the residence time of the transcription factor at the promoter site, which is surprising since transcription can persist on the time scale of hours. This finding raises questions about the role of rapid exchange in the mechanism and regulation of transcription. It suggests that the FRAP techniques being developed will lead to improved understanding of these complex intracellular processes.