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

项目摘要 利用RNA聚合酶(RNAP)合成RNA，并将其折叠成具有生物功能三种类型的RNA. 空间形状是齐头并进的。RNA合成的动力学决定了折叠的结果，并 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合成被依赖于序列的停顿中断，在此期间，聚合酶 保持与核酸的结合而不发生活跃的核苷酸加成。这些暂停创建窗口 转录调控发生的时间。我们的研究计划将在原子水平上解决停顿 停顿的机制和对共转录事件的贡献，如RNA的折叠。第一个方向 该计划的目标是开发工具来捕获和可视化转录过程中的RNA折叠中间产物 并了解pH对RNAP合成RNA的动力学的影响，从而了解RNA折叠途径。 由此产生的工具将引起RNA社区的广泛兴趣，因为它们可以用于跟踪折叠 其他具有重要生物学意义的RNA。第二个方向将应用这些工具来绘制共转录RNA “健康”和突变的人类线粒体转移RNA(mt-tRNA)的折叠差异，从而提供 致病的mt-tRNA突变的结构基础。此外，我们将评估以下方面的贡献 线粒体RNAP暂停到未突变和疾病变异的mt-tRNA的差异折叠，因此 扩大转录暂停在这个关键细胞器中发挥的调节作用的武器库。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 是为了迎合不断变化的蜂窝对能源的需求。拟议研究的完成情况 将对理解基因表达的基本原理以及 在合成生物学和与线粒体DNA相关的疾病的分子机制方面的应用。

Project Summary The synthesis of RNA by RNA polymerase (RNAP) and folding of that RNA into its biologically functional three- dimensional shape go hand in hand. The kinetics of RNA synthesis determine the folding outcome, and 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 the polymerase 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 at the atomic level the pausing mechanisms and contribution of pausing to co-transcriptional events, such as folding of RNA. 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. The second direction will apply those tools to map co-transcriptional RNA folding differences in “healthy” and mutated human mitochondrial transfer RNAs (mt-tRNA), thus providing the structural basis for disease-causing mt-tRNA mutations. Additionally, we will assess the contribution of mitochondrial RNAP pausing to the differential folding of unmutated and disease-variant mt-tRNA, thus expanding the arsenal of regulatory roles transcriptional pausing plays in this key organelle. Finally, the third research direction will address how the balance between transcription of mitochondrial DNA 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 the basic principles of gene expression, as well as to the applications in synthetic biology and to the molecular mechanisms of diseases linked to mitochondrial DNA.