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的，也不知道它们与什么结合。我们的工作集中在使用荧光恢复后的光漂白（FRAP），以调查在特定的启动子位点，也在整个细胞核的其他通用网站的转录因子的流动性。我们先前已经在小鼠细胞系中显示，GFP标记的糖皮质激素受体与特定启动子结合最多60秒，即使转录持续数小时。为了获得一个精确的估计多久糖皮质激素受体保持绑定的启动子，我们已经开发了数学模型来分析的扩散和结合的相互作用的受体发生在FRAP实验。我们的模型预测，个别糖皮质激素受体结合在启动子不到一秒钟。这种非常短暂的结合提出了新的问题，即如何在转录因子如此短的停留时间内组装转录复合物。糖皮质激素受体结合的前述分析是使用包含2Mb DNA的200个启动子和报告基因的合成串联阵列进行的。可能是这个人造系统的产物为了解决这个问题，我们在一个完全不同的生物体中开发了一个完全自然的系统。我们正在使用一个小的（2 kb）阵列的10个基因，自然出现在常见的酵母菌株。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进行FRAP。我们发现，这种转录因子也被绑定到其启动子瞬时，这表明这种瞬时的相互作用不是一个文物，可能是相当普遍的。

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 (FRAP) 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 FRAP 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.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.