Transcription: Mechanism and Regulation

项目概要/摘要 这项工作旨在了解基因表达如何在转录水平上控制 RNA聚合酶（RNAP）活性的改变。大肠杆菌RNAP作为一种 转录机制研究的特殊模型系统和理解基因的范例 在细菌中表达。细菌生活在复杂多变的环境中，需要基因表达被 对生长状态或环境条件的变化高度敏感。给定基因的表达是 由DNA模板序列、调节蛋白和反应的贡献决定。 条件虽然由DNA序列提供的输入是“硬编码”的（除非突变改变），但输入 调节因子和反应条件是灵活的，使它们能够响应变化， 环境条件的改变。许多转录因子和反应条件可以 调节基因表达。因此，基因表达的变化是对生长变化的反应， 状态或环境条件不能仅从DNA序列先验地预测。实验 建议将使系统级描述的序列依赖，因素依赖，条件- 依赖的转录景观。转录的每个步骤都可以是速率限制的，并且 从而成为监管的潜在目标。在项目期间，转录控制机制 在初始转录和延伸期间，即，RNA合成发生的阶段，将被研究。在 在今后的工作中，研究将扩大到包括启动和终止，使用类似的方法。一个显著 所描述的工作的一个特点是开发了监测广泛的转录的方法。 序列空间，在体外和体内。此外，使用E. coli RNAP作为模型 系统，能够直接分析任何转录因子或反应条件对 RNAP活性，在体外和体内。RNAP活动的经验测量结果， 广泛的序列空间或整个E.大肠杆菌基因组将整合到一个位点和碱基特异性 描述RNAP活性如何由DNA序列、转录、 因素和条件。所获得的生物学知识将提供一个前所未有的丰富的描述， E.并为将这种方法扩展到其他 生物，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.