Probing Transcriptional Activation at the Molecular Level

描述(由申请人提供)：拟议研究的广泛目标是使用一套化学方法在分子水平上了解转录激活剂及其目标蛋白在转录机制中的相互作用。转录调控不当是人类疾病的原因或结果，如癌症、代谢紊乱和传染病。因此，抑制或促进调节转录的蛋白质-蛋白质相互作用的分子既是机械探针，从长远来看，也是纠正转录错误的治疗剂。然而，只有少数这样的分子被报道，部分原因是对转录调控机制的了解仍然有限。在转录激活物的情况下，蛋白质以信号响应的方式激活特定基因的转录，对于转录机制中的相互作用伙伴或功能相关的热力学参数，人们几乎不知道。作为特定目标1的一部分，我们将从动力学角度描述几个转录激活子与它们的目标蛋白之间的相互作用，以解析基因上调水平与激活子-转录机械蛋白复合体的结合和解离速率之间的关系。在特定的AIMS 2和3(酿酒酵母)中，将通过体内交联实验和交联产物的FT-ICR质谱分析来确定转录激活子的生理相关靶点，从而解决真核转录中一个长期存在的基本机制问题。综上所述，通过这些实验产生的数据将提供一个高分辨率的地图，显示转录激活剂在启动基因上调时，在转录机制中结合了什么以及在哪里。这些信息将极大地有助于发现以转录为靶标的小分子，并从长远来看，有助于开发以单个转录激活子-转录机械蛋白相互作用为靶标的小分子，这些小分子与人类疾病有关。公共卫生相关性：这项提案中概述的实验将提供转录激活剂结合情况的详细图景，以及个别结合事件对转录调控的贡献。这些数据将使设计更有效的小分子筛选成为可能，这些小分子起到转录激活剂或抑制物的作用，此外，还将提供一种快速评估特异性的策略。最终，这将促进基于转录的疗法的发现和发展。

DESCRIPTION (provided by applicant): The broad objective of the proposed research is to obtain a molecular-level understanding of the interactions between transcriptional activators and their target proteins within the transcriptional machinery using a suite of chemical approaches. Misregulation of transcription is either a cause or an effect of human diseases such as cancer, metabolic disorders, and infectious diseases. Thus, molecules that inhibit or promote the protein-protein interactions that regulate transcription are highly desirable both as mechanistic probes and, in the long term, as therapeutic agents that correct transcriptional errors. Only a handful of such molecules have been reported, however, due in part to the still-limited understanding of the mechanism of transcriptional regulation. In the case of transcriptional activators, proteins that activate the transcription of particular genes in a signal- responsive fashion, little conclusive is known about either the interaction partners within the transcriptional machinery or the functionally relevant thermodynamic parameters. As part of Specific Aim 1, we will kinetically characterize the interactions between several transcriptional activators and their target proteins in order to parse the connection between levels of gene up-regulation and the rates of association and dissociation of activator-transcriptional machinery protein complexes. In Specific Aims 2 and 3 in vivo (S. cerevisiae) crosslinking experiments along with FT-ICR mass spectrometric analysis of crosslinking products will be employed to identify the physiologically relevant targets of transcriptional activators, thus addressing a long-standing and fundamental mechanistic question in eukaryotic transcription. Taken together, the data generated through these experiments will provide a high resolution map of what and where transcriptional activators bind in the transcriptional machinery as they initiate gene up-regulation. This information will be enormously enabling for the discovery of transcription-targeting small molecules and, longer term, for the development of small molecules that target individual transcriptional activator-transcriptional machinery protein interactions implicated in human disease. PUBLIC HEALTH RELEVANCE: The experiments outlined in this proposal will provide a detailed picture of the binding profile of transcriptional activators and the contribution that the individual binding events make to transcriptional regulation. These data will make it possible to design more effective screens of small molecules that function as activators or inhibitors of transcription and, further, will provide a strategy by which specificity can be quickly evaluated. Ultimately, this will facilitate the discovery and development of transcription-based therapeutics.