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

细菌和人类线粒体的转录机制及相关基因表达过程

• 批准号: 10810460
• 负责人: Tatiana Vladimirovna Mishanina
• 金额: $ 0.76万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10810460
• 关键词: 3-Dimensional; Bacteria; Binding; Cells; Communities; DNA; DNA Sequence; DNA-Directed RNA Polymerase; Diagnostic; Disease; Engineering; Equilibrium; Event; Gene Expression; Gene Expression Process; Genetic Transcription; Human; Interruption; Kinetics; Link; Maps; Microbe; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial RNA; Molecular; Mutate; Mutation; Nucleic Acids; Nucleotides; Organelles; Organism; Outcome; Pathway interactions; Play; Polymerase; Process; Proteins; RNA; RNA Folding; RNA chemical synthesis; Research; Role; Shapes; Speed; Temperature; Therapeutic; Time; Transcriptional Regulation; Transfer RNA; Variant; Visualization; insight; interest; novel; programs; small molecule; synthetic biology; tool

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.

项目概要 通过 RNA 聚合酶 (RNAP) 合成 RNA，并将该 RNA 折叠成具有生物功能的三层结构： RNA 合成的动力学决定了折叠结果，并且是密切相关的。 受多种因素影响，如细胞内温度、pH、小分子浓度和 细胞中的蛋白质，以及转录为 RNA 的 DNA 的确切序列并不是连续的。 过程：RNAP 的 RNA 合成被序列依赖性暂停打断，在此期间聚合酶 保持与核酸结合，而不发生活性核苷酸添加。这些暂停产生了窗口。 我们的研究计划将在原子水平上解决转录调节的时间问题。 暂停对共转录事件（例如 RNA 折叠）的机制和贡献 第一个方向。 该项目旨在开发用于捕获和可视化转录过程中 RNA 折叠中间体的工具 并了解 pH 对 RNAP 合成 RNA 动力学以及 RNA 折叠途径的影响。 由此产生的工具将引起 RNA 界的广泛兴趣，因为它们可用于跟踪折叠 第二个方向将应用这些工具来绘制共转录 RNA 的图谱。 折叠“健康”和突变的人类线粒体转移 RNA (mt-tRNA) 的差异，从而提供 此外，我们将评估致病 mt-tRNA 突变的结构基础。 线粒体 RNAP 暂停未突变和疾病变异 mt-tRNA 的差异折叠，因此 扩大转录暂停在这个关键细胞器中的调控作用最后是第三个。 研究方向将解决线粒体DNA转录与其包装之间的平衡 目的是为了满足不断变化的细胞对能量的需求。完成拟议的研究。 将彻底改变对基因表达基本原理的理解，以及 合成生物学中的应用以及与线粒体 DNA 相关的疾病的分子机制。