Transcription factor mobility

蛋白质在细胞核中移动，并与那里的结合位点短暂地相互作用，但在大多数情况下，我们不知道它们为什么如此移动，也不知道它们与什么结合在一起。我们的工作重点是利用光漂白后的荧光恢复和荧光相关光谱来研究转录因子在特定启动子位点和整个细胞核中其他一般位点的迁移性。我们之前在小鼠细胞系中表明，gfp标记的糖皮质激素受体在特定启动子上结合最多60秒，即使转录持续数小时。为了准确估计糖皮质激素受体与启动子结合的时间，我们建立了数学模型来分析光漂白实验后荧光恢复过程中受体的扩散和结合相互作用。我们的模型预测单个糖皮质激素受体在启动子上结合的时间不到一秒。这种非常短暂的结合提出了新的问题，即转录因子如何在如此短的停留时间内组装转录复合体。同时，我们已经表明，光漂白后荧光恢复的不同分析程序可以产生不同的停留时间估计。这表明，在估计活细胞结合参数方面仍存在一些不确定性，需要开发替代测量程序以达到共识估计。为了使活细胞中糖皮质激素受体结合的可视化，前面的糖皮质激素受体结合分析是使用包含2Mb DNA的200个启动子和报告基因的合成串联阵列进行的。这就提出了一个问题，即检测到的瞬时结合是否可能是该人工系统的产物。为了解决这个问题，我们在一个完全不同的有机体中开发了一个完全自然的系统。我们使用的是一个由十个基因组成的小阵列（2kb），这些基因是在普通酵母菌株中自然产生的。通过用GFP标记相关的转录因子，我们已经能够可视化该阵列并对转录因子执行FRAP。我们发现这种转录因子也会短暂地与其启动子结合，这表明这种短暂的相互作用不是人为的，而且可能相当普遍。

Proteins move in the nucleus and transiently interact with binding sites there, but in most cases we do not know why they are so mobile or what they are bound to. Our work has focused on using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy to investigate the mobility of transcription factors both at specific promoter sites and also at other generic sites throughout the nucleus. We have previously shown in a mouse cell line that the GFP-tagged glucocorticoid receptor is bound at a specific promoter for at most 60 seconds, even though transcription persists for several hours. To obtain a precise estimate of how long the glucocorticoid receptor remains bound to the promoter, we have developed mathematical models to analyze the diffusion and binding interactions of the receptor that occur during the fluorescent recovery after photobleaching experiment. Our model predicts that individual glucocorticoid receptors are bound at the promoter for less than a second. This very transient binding raises new questions about how the transcription complex can be assembled with such short residence times of the transcription factor. At the same time, we have shown that different analysis procedures for fluorescence recovery after photobleaching can yield different estimates of residence times. This shows that there are still some uncertainties in the estimation of live cell binding parameters that will require developing alternate measurement procedures to arrive at consensus estimates. To enable visualization of glucocorticoid receptor binding in live cells, the preceding analyses of glucocorticoid receptor binding were performed using a synthetic tandem array of 200 promoters and reporter genes comprising 2Mb of DNA This has raised the question of whether the transient binding detected might be an artifact of this artificial system. To address this, we have developed a completely natural system in a completely different organism. We are using a small (2 kb) array of ten genes that arises naturally in common yeast strains. By tagging the associated transcription factor with GFP, we have been able to visualize this array and perform FRAP on the transcription factor. We find that this transcription factor is also bound transiently to its promoter, suggesting that such transient interactions are not an artifact and may be rather common.