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：
7466227
负责人：
ANNA K. MAPP
金额：
$ 25.85万
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 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7466227
关键词：
Activator Appliances Address Affinity Amino Acids Binding Binding Sites Biochemical Biochemical Genetics Chemicals Chromatin Class Communicable Diseases Communities Complex Conflict (Psychology)Coupled Cues DNA Binding Data Development Disputes Dissociation Equilibrium Eukaryota Eukaryotic Cell Event Formaldehyde Freezing Funding Genes Genetic Genetic Transcription 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 Public Health RNA Polymerase II Rate Recruitment Activity Reporter Genes Reporting Research Resolution Role Saccharomyces cerevisiae Series Signal Transduction Specificity System TP53 gene 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 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通过使用一系列化学方法来获得对转录机械中转录激活剂及其靶蛋白之间相互作用的分子级别的理解。转录的正调是癌症，代谢性疾病和传染病等人类疾病的原因或影响。因此，抑制或促进调节转录的蛋白质蛋白相互作用的分子既可以作为机械探针，又是纠正转录误差的治疗剂。但是，仅报道了少数此类分子，部分原因是对转录调控机制的仍然有限的理解。在转录激活剂的情况下，以信号响应方式激活特定基因转录的蛋白质对转录机械中的相互作用伙伴或功能相关的热力学参数知之甚少。作为特定目标1的一部分，我们将在动力学上表征几种转录激活剂与其靶蛋白之间的相互作用，以解析基因上调水平上调与激活剂转录机械蛋白蛋白复合物的关联速率和解离之间的联系。在特定的目标2和3中，将采用交联实验（酿酒酵母）的交联实验以及交联产物的FT-ICR质谱分析，以识别转录激活剂的生理相关目标，从而解决了真生性转录的长期和基本机械性问题。综上所述，通过这些实验生成的数据将提供一个高分辨率图，即转录激活因子在转录机械中启动基因上调时在转录机械中结合的位置。这些信息将非常有助于发现靶向转录的小分子，从长远来看，对于针对人类疾病涉及的个体转录激活剂转录机械蛋白相互作用的小分子的发展。公共卫生相关性：本提案中概述的实验将提供转录激活剂的结合曲线以及单个结合事件对转录调节的贡献的详细图片。这些数据将使设计更有效的小分子的筛选，这些筛选起着激活剂或转录抑制剂的作用，此外，将提供一种可以快速评估特异性的策略。最终，这将有助于基于转录的治疗剂的发现和开发。