Transcription factor mobility

蛋白质在细胞核内移动，并与那里的结合部位短暂地相互作用，但在大多数情况下，我们不知道它们为什么如此可移动或它们与什么结合。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置和整个细胞核中其他普通位置的迁移率。我们之前已经在小鼠细胞系中表明，GFP标记的糖皮质激素受体与特定启动子结合的时间最长为60秒，即使转录持续几个小时。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作用。我们的模型预测，单个糖皮质激素受体与启动子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，即如何在转录因子的停留时间如此短的情况下组装转录复合体。同时，我们已经证明，对光漂白后的荧光恢复的不同分析方法可以产生不同的停留时间估计。这表明在活细胞结合参数的估计中仍然存在一些不确定性，这将需要开发替代测量程序来获得一致的估计。为了能够在活细胞中可视化糖皮质激素受体结合，前面的糖皮质激素受体结合分析是使用由200个启动子和报告基因组成的合成串联阵列进行的，该阵列包括2Mb的DNA。这提出了一个问题，即检测到的瞬时结合是否可能是该人工系统的人工制品。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。我们正在使用一个由10个基因组成的小阵列(2kb)，这些基因自然出现在常见的酵母菌株中。通过用GFP标记相关的转录因子，我们已经能够可视化这个阵列并在转录因子上进行FRAP。我们发现，该转录因子也与其启动子瞬时结合，这表明这种瞬时相互作用不是人工产物，可能相当常见。

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.