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Probing Transcriptional Activation at the Molecular Level

在分子水平上探测转录激活

基本信息

批准号：
8065354
负责人：
ANNA K. MAPP
金额：
$ 25.97万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-04-01 至 2013-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8065354
关键词：
Activator Appliances Address Affinity Amino Acids Binding Binding Sites Biochemical Chemicals Chromatin Communicable Diseases Communities Complex Conflict (Psychology)Coupled Cues DNA Binding Data Development Disputes Dissociation Equilibrium Eukaryota Event Formaldehyde Freezing Funding Gene Activation Genes Genetic Genetic Transcription Health In Vitro Individual Light Link Location Malignant Neoplasms Maps Measures Mediator of activation protein Metabolic Diseases Modeling Molecular Mutagenesis Pattern Plasmids Processed Genes Protein Binding Proteins RNA Polymerase II Recruitment Activity Reporter Genes Reporting Research Resolution Role Saccharomyces cerevisiae Series Signal Transduction Specificity System Tertiary Protein Structure Testing Therapeutic Therapeutic Agents Thermodynamics Time TimeLine Transcription Coactivator Transcriptional Activation Transcriptional Regulation Up-Regulation VP 16 Western Blotting base chromatin immunoprecipitation covalent bond crosslink design human NCYM protein human disease in vivo inhibitor/antagonist irradiation promoter protein complex protein protein interaction research study response small molecule

项目摘要

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.

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转录机制内获得转录激活因子及其靶蛋白之间相互作用的分子水平理解。转录失调是人类疾病如癌症、代谢紊乱和传染病的原因或结果。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作为机制探针和长期作为校正转录错误的治疗剂都是非常理想的。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理解仍然有限。在转录激活因子（以信号响应方式激活特定基因转录的蛋白质）的情况下，关于转录机制内的相互作用伴侣或功能相关的热力学参数几乎没有定论。作为特定目标1的一部分，我们将从动力学上表征几种转录激活因子及其靶蛋白之间的相互作用，以解析基因上调水平与激活因子-转录机器蛋白复合物的结合和解离速率之间的联系。在特定目的2和3体内（S.酿酒酵母）交联实验沿着交联产物的FT-ICR质谱分析将用于鉴定转录激活因子的生理学相关靶标，从而解决真核转录中长期存在的基本机制问题。总之，通过这些实验产生的数据将提供一个高分辨率的地图，什么和转录激活因子结合在转录机制，因为他们启动基因上调。这些信息将极大地促进转录靶向小分子的发现，并且从长远来看，促进靶向与人类疾病有关的个体转录激活因子-转录机制蛋白相互作用的小分子的开发。公共卫生相关性：在这个建议中概述的实验将提供一个详细的图片的结合概况的转录激活因子和个人的结合事件的转录调控的贡献。这些数据将使设计更有效的小分子筛选成为可能，这些小分子可以作为转录的激活剂或抑制剂，并且还将提供一种可以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开发。