Transcription Factor Mobility

采用光漂白后荧光恢复法（FRAP）研究了gfp标记的糖皮质激素受体（GFP-GR）在体外培养腺癌3617细胞细胞核内的迁移率。GFP-GR是一种蛋白质转录因子，是小鼠乳腺肿瘤病毒（MMTV）细胞核DNA启动子位点的配体。为了定量解释FRAP测量结果，建立了数学模型来描述GFP-GR的核内结合和扩散对荧光恢复的影响。这些模型包括与分布在整个细胞核中的位点的低亲和力非特异性结合，以及与离散的启动子位点阵列的高亲和力结合。这些模型考虑到原子核的有限体积。利用有限元软件在两个空间维度上得到控制方程的数值解，以适应高亲和力位点的空间定位。通过与一维解析解的仿真对比，验证了二维数值解的正确性。参数灵敏度分析表明，忽略扩散会在绑定参数估计中产生严重的误差。这些模型被应用于GFP-GR的FRAP测量，其中高亲和力结合区代表了重复MMTV启动子靶点的局部阵列。一个结果是转录因子在启动子位点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技术将有助于提高对这些复杂细胞内过程的理解。

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.