Transcription: Mechanism and Regulation

项目概要/摘要 拟议的工作旨在了解转录水平上的基因表达控制 RNA 聚合酶 (RNAP) 活性改变的结果。大肠杆菌 RNAP 作为 用于转录机制研究和理解基因的范例的特殊模型系统 在细菌中表达。细菌生活在复杂、波动的环境中，需要基因表达 对生长状态或环境条件的变化高度敏感。给定基因的表达量为 由 DNA 模板序列、调节蛋白和反应的贡献决定 条件。虽然 DNA 序列提供的输入是“硬编码的”（除非发生突变），但输入 调节因素和反应条件提供的灵活性，使其能够响应变化 环境条件的变化。多种转录因子和反应条件可以 调节基因表达。因此，基因表达的变化是响应生长变化而发生的。 仅凭 DNA 序列无法先验地预测状态或环境条件。实验 所提出的将能够对序列相关、因素相关、条件相关的系统级描述成为可能。 大肠杆菌中的依赖性转录景观。转录的每个步骤都可以进行速率限制，并且 从而成为潜在的监管目标。项目期间，转录控制机制 将研究初始转录和延伸期间，即发生 RNA 合成的阶段。在 在未来的工作中，研究将使用类似的方法扩展到包括启动和终止。一个有特色的 所描述的工作的特点是开发了广泛监测转录的方法 体外和体内的序列空间。此外，使用大肠杆菌 RNAP 作为模型提供了灵活性 系统，可以直接分析任何转录因子或反应条件的贡献 RNAP 活性，体外和体内。 RNAP 活性的经验测量结果 广泛的序列空间或整个大肠杆菌基因组将被整合到位点和碱基特异性 定量模型描述 RNAP 活性如何通过 DNA 序列、转录的输入来指定 因素、条件。获得的生物学知识将为生物提供前所未有的丰富描述。 大肠杆菌中基因表达控制的体系结构，并为将该方法扩展到其他方法提供基础 生物体，包括真核生物。

PROJECT SUMMARY/ABSTRACT The work proposed is designed to understand how gene expression control at the transcriptional level results from alterations in the activity of RNA polymerase (RNAP). Escherichia coli RNAP serves as an exceptional model system for mechanistic studies of transcription and as a paradigm for understanding gene expression in bacteria. Bacteria live in complex, fluctuating environments requiring gene expression to be highly responsive to changes in growth state or environmental conditions. The expression of a given gene is determined by contributions from the sequence of the DNA template, regulatory proteins, and reaction conditions. While the input provided by DNA sequence is “hard-coded” (barring mutational change), the inputs provided by regulatory factors and reaction conditions are flexible, enabling them to change in response to alterations in environmental conditions. A multitude of transcription factors and reaction conditions can modulate gene expression. Thus, changes in gene expression occurring in response to alterations in growth state or environmental conditions cannot be predicted a priori from DNA sequence alone. Experiments proposed will enable a systems-level description of the sequence-dependent, factor-dependent, condition- dependent transcriptional landscape in Escherichia coli. Each step of transcription can be rate limiting, and thus serve as a potential target of regulation. During the project period, mechanisms of transcriptional control during initial transcription and elongation, i.e., the stages when RNA synthesis occurs, will be investigated. In future work, studies will expand to include initiation and termination, using similar approaches. A distinguishing feature of the work described has been the development of methods to monitor transcription across extensive sequence space, both in vitro and in vivo. In addition, the flexibility provided by use of E. coli RNAP as a model system, enables straightforward analysis of the contributions of any transcription factor or reaction condition to RNAP activity, both in vitro and in vivo. The results of empirical measurements of RNAP activity across extensive sequence space or across the entire E. coli genome will be integrated into a site- and base-specific quantitative model describing how RNAP activity is specified by inputs from DNA sequence, transcription factors, and conditions. The biological knowledge gained will provide an unprecedently rich description of the architecture of gene expression control in E. coli and provide the basis for extending this approach to other organisms, including eukaryotes.