Transcription factor mobility

转录因子迁移率

• 批准号: 7338724
• 负责人: JAMES M MCNALLY
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7338724
• 关键词: Transcription; factor; mobility

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.

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或它们与什么结合。我们的工作重点是使用光漂白后的荧光恢复（FRAP）来研究转录因子在特定启动子位点以及整个细胞核中其他通用位点的迁移性。我们之前已经在小鼠细胞系中证明，尽管转录持续了几个小时，但 GFP 标记的糖皮质激素受体在特定启动子上的结合时间最多为 60 秒。为了精确估计糖皮质激素受体与启动子结合的时间，我们开发了数学模型来分析 FRAP 实验期间发生的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的结合时间不到一秒。这种非常短暂的结合提出了新的问题，即转录复合物如何在如此短的转录因子停留时间下组装起来。为了实现活细胞中糖皮质激素受体结合的可视化，之前的糖皮质激素受体结合分析是使用合成串联阵列进行的包含 2Mb DNA 的 200 个启动子和报告基因 这引发了一个问题：检测到的瞬时结合是否可能是该人工系统的人为产物。为了解决这个问题，我们在完全不同的有机体中开发了一个完全自然的系统。我们使用的是常见酵母菌株中自然产生的十个基因的小型（2 kb）阵列。通过用 GFP 标记相关的转录因子，我们能够可视化该阵列并对转录因子执行 FRAP。我们发现该转录因子也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，并且可能相当常见。