Structural and biophysical studies of proteins, nucleic acids, and their complexes

项目摘要 这项提议的中心主题是核酸、蛋白质和 他们的互动。作为我们未来研究的重点的两大主题是：1)结构和 拓扑异构酶的作用机制，以及2)非编码RNA分子的结构和作用机制，特别是 T-盒核糖开关。我们工作的目的是发现和探索有助于阐明 生物分子的结构和机理。 拓扑异构酶是一种普遍存在的蛋白质，存在于生命的所有三个领域。他们参与了 几个细胞过程及其细胞作用的重要性被以下事实所强调：它们是 几种癌症化疗药物和抗生素的靶标。对其结构、机制和功能的研究 拓扑异构酶的功能不仅加深了我们在许多不同水平上相互作用的蛋白质的理解 并改变其拓扑性质，也为辅助设计DNA提供了重要信息 新的治疗剂。我们将继续对拓扑异构酶进行全面的研究，重点是 三个大型和互补的子项目：一)IA型拓扑异构酶的单分子研究，二)结构 天然底物上拓扑异构酶的研究，以及III)旋转酶与小分子拓扑异构酶络合物的结构研究 圈住DNA。这些子项目将帮助回答将该领域推进到下一个阶段所需的关键问题 水平。 核糖开关是在mRNAs的非翻译区发现的配体特异的顺式调节RNA元件 对特定的外部信号做出反应，从而影响下游基因的转录或翻译。细菌T- 盒状核糖开关代表了一类独特的核糖开关，它不结合小分子配体，相反，它们 识别和结合tRNA分子，并直接检测其氨基酰化状态。他们代表着一位出色的 理解RNA结构和体系结构的范式，其他RNA分子对RNA分子的识别， 以RNA为基础的基因调控，以及古代RNA世界中基因调控功能的进化。我们会 继续和扩大我们对T-box核糖开关的研究，集中在三个子项目上：i)单分子 翻译T盒核糖开关的研究，II)典型T盒的结构和单分子研究 3)串联T-box核糖开关的结构研究。这三个子项目将推进 我们对T盒核糖开关和非编码RNA分子的理解，这些分子仍然不完全 在原子水平上被理解。

Project Summary The central theme of this proposal is structural and mechanistic studies of nucleic acids, proteins, and their interactions. The two broad topics that will serve as foci for our future research are: 1) the structure and mechanism of topoisomerases, and 2) the structure and mechanism of non-coding RNA molecules, particularly T-box riboswitches. The aim of our work is to discover and explore paradigms that help to illuminate the structure and mechanism of biological molecules. Topoisomerases are ubiquitous proteins found across all three domains of life. They are involved in several cellular processes and the importance of their cellular role is underscored by the fact that they are the target of several cancer chemotherapeutic agents and antibiotics. The study of the structure, mechanism, and function of topoisomerases not only furthers our understanding at many different levels of proteins that interact with DNA and alter its topological properties, but also provides important information to aid in the design of new therapeutic agents. We will continue our comprehensive studies of topoisomerases with an emphasis on three large and complementary sub-projects: i) Single molecule studies of type IA topoisomerases, ii) Structural studies of topoisomerases on natural substrates, and iii) Structural studies of gyrase in complex with mini- circle DNA. These sub-projects will help answer crucial questions that are needed to advance the field to the next level. Riboswitches are ligand-specific cis-regulatory RNA elements found in untranslated regions of mRNAs that respond to defined external signals to affect transcription or translation of downstream genes. Bacterial T- box riboswitches represent a unique class of riboswitches that do not bind small molecule ligands, instead they recognize and bind tRNA molecules and sense directly their aminoacylation state. They represent an excellent paradigm to understand RNA structure and architecture, recognition of RNA molecules by other RNA molecules, RNA-based gene regulation, and the evolution of gene regulatory function from the ancient RNA world. We will continue and expand our studies of T-box riboswitches by concentrating in three subprojects: i) Single molecule studies of translational T-box riboswitches, ii) Structural and single molecule studies of typical T-box riboswitches, and iii) Structural studies of tandem T-box riboswitches. These three sub-projects will advance our understanding of T-box riboswitches and non-coding RNA molecules, molecules that are still not fully understood at the atomic level.