PROTEIN/PROTEIN INTERACTIONS OF CREB/ATF PROTEINS

信号转导通路最终汇聚在 转录激活以产生特定的基因表达模式 对环境刺激的反应。转录的启动 通过这些信号通路的调节受坐标的调节 结合特定转录因子的表达和/或激活 到基因的控制区。对机制的具体见解 潜在的转录激活最近出现在这些研究中。 这些转录因子的结构和功能。这个 CREB/ATF转录反式激活蛋白家族只有 最近被发现，并似乎提供了 细胞激活剂对基因表达的调控作用 信号通路与病毒对基因表达的调控 反式激活蛋白。理解的性质和重要性 真核基因机制中的蛋白质/蛋白质相互作用 表达是理解正常和异常调控的关键 基因转录的结果。我们最近开发了一种快速而灵敏的 允许检测这些相互作用的技术 哺乳动物转录因子。利用~(125)I标记的重组CREB 和ATF-2多肽来探测HeLa核提取物的蛋白质印迹，我们有 确定了多个单独的核因素，形成了特定的 含亮氨酸拉链的蛋白质/蛋白质相互作用 转录调节蛋白。在本申请中，我们建议 利用这一技术分离表达蛋白质的cDNA克隆 与CREB和/或ATF-2的特异性蛋白质/蛋白质相互作用。此外, 我们将对DNA结合和转录进行详细分析 几种CREB/ATF蛋白的特性蛋白质/蛋白质的测定 我们开发的交互将允许系统地 真核生物调控相关蛋白的鉴定和克隆 转录调控。金黄色葡萄球菌的鉴定与表征 真核转录复合体的组成将允许研究 阐述细胞正常和异常控制的分子机制 基因表达。

Signal transduction pathways converge ultimately at the level of transcriptional activation to produce specific patterns of gene expression in response to environmental stimuli. The initiation of transcription mediated by these signalling pathways is regulated by the coordinate expression and/ or activation of specific transcription factors that bind to the control regions of genes. Specific insights into the mechanisms underlying transcriptional activation have recently arisen from the studies of the structure and functions of these transcription factors. The CREB/ATF family of transcriptional transactivating proteins has only recently been discovered and appears to provide a link between the regulation of gene expression in response to activators of cellular signalling pathways and the regulation of gene expression by viral transactivating proteins. Understanding the nature and importance of protein/protein interactions in the mechanisms of eukaryotic gene expression is essential to understanding the normal and aberrant regulation of gene transcription. We have recently developed a rapid and sensitive technique which allows the detection of these interactions between mammalian transcription factors. Utilizing 125I-labelled recombinant CREB and ATF-2 peptides to probe western blots of HeLa nuclear extracts, we have identified multiple separate nuclear factors that form specific protein/protein interactions with these leucine zipper-containing transcriptional regulatory proteins. In this application, we propose to utilize this technique to isolate cDNA clones expressing proteins that form specific protein/protein interactions with CREB and/or ATF-2. In addition, we will perform detailed analyses of the DNA-binding and transcriptional characteristics of several CREB/ATF proteins. The assay of protein/protein interactions we have developed will allow a systematic approach to identifying and cloning proteins involved in the control of eukaryotic transcriptional regulation. The identification and characterization of the components of eukaryotic transcription complexes will allow studies that address the molecular mechanisms of normal and aberrant control of cellular gene expression.