Mechanism of transcription and related gene expression processes in bacteria and human mitochondria

项目摘要 在细胞中，由RNA聚合酶(RNAP)产生的RNA折叠成其功能三维形状，而它 正在由RNAP合成。决定RNA折叠结果的RNA合成动力学是 受到多种因素的影响，如细胞内温度、pH、小分子浓度和 细胞中的蛋白质，以及DNA转录成RNA的准确序列。抄写不是连续的 过程：RNAP的RNA合成被顺序相关的暂停中断，在此期间RNAP保持 与核酸结合而不发生活跃的核苷酸加成。这些停顿创造了时间窗口 转录的调节才能发生。我们的研究计划将解决在 原子水平和暂停对共转录事件的贡献，如细菌和 人类线粒体。该计划的第一个方向是开发捕获和可视化RNA的工具 在转录过程中折叠中间产物，并了解pH对RNA合成动力学的影响 RNAP，从而RNA折叠途径。由此产生的工具将引起RNA社区的广泛兴趣 因为它们可以用来跟踪其他生物重要RNA的折叠。第二个方向将适用 用于绘制“健康”和突变人类共转录RNA折叠差异的工具 线粒体转移RNAs(mt-tRNA)，从而为致病mt-tRNA提供结构基础 突变。我们将评估线粒体RNAP(MtRNAP)暂停对差异折叠的贡献 未突变与疾病变异型mt-tRNA的比较。此外，我们还将测试mtRNAP的另一个假设功能 暂停：线粒体DNA(MtDNA)转录与其复制的偶联，这对维持 足够的线粒体DNA拷贝来生产氧化磷酸化机制的蛋白质成分。最后， 第三个研究方向将解决线粒体DNA转录和包装之间的平衡是如何 实现了满足不断变化的蜂窝对能源的需求。建议的研究完成后， 对基因表达的基本原理的理解具有革命性，分子 与线粒体DNA相关的疾病机制，以及基于RNA的工具在合成生物学中的应用。

Project Summary In the cell, the RNA made by RNA polymerase (RNAP) folds into its functional three-dimensional shape while it is being synthesized by RNAP. The kinetics of RNA synthesis, which determine the RNA folding outcome, are influenced by myriad factors, such as intracellular temperature, pH, concentrations of small molecules and proteins in the cell, and the exact sequence of DNA being transcribed into RNA. Transcription is not a continuous process: RNA synthesis by RNAP is interrupted by sequence-dependent pauses, during which RNAP remains bound to the nucleic acids without active nucleotide addition occurring. These pauses create windows of time for regulation of transcription to occur. Our research program will address the mechanisms of pausing at the atomic level and the contribution of pausing to co-transcriptional events, such as folding of RNA, in bacteria and human mitochondria. The first direction of the program aims to develop tools for capturing and visualizing RNA folding intermediates during transcription and to understand the effect of pH on the kinetics of RNA synthesis by RNAP, and thus the RNA folding pathway. The resulting tools will be of broad interest to the RNA community because they can be applied to follow folding of other biologically important RNAs. A second direction will apply those tools to map the differences in co-transcriptional RNA folding of “healthy” and mutated human mitochondrial transfer RNAs (mt-tRNA), thus providing the structural basis for disease-causing mt-tRNA mutations. We will assess the contribution of mitochondrial RNAP (mtRNAP) pausing to the differential folding of unmutated vs. disease-variant mt-tRNA. Additionally, we will test another hypothesized function of mtRNAP pausing: coupling of transcription of mitochondrial DNA (mtDNA) to its replication, which is critical for maintaining enough mtDNA copies for production of protein components of the oxidative phosphorylation machinery. Finally, a third research direction will address how the balance between transcription of mtDNA and its packaging is achieved to cater to the ever-changing cellular needs for energy. The completion of the proposed research will be transformative to the understanding of basic principles governing gene expression, the molecular mechanisms of diseases linked to mtDNA, and to the applications of RNA-based tools in synthetic biology.