Catalysis and Cooperativity in Regulatory RNAs

描述(由申请人提供)：项目摘要：RNA曾经被认为是遗传信息流中的被动中间体，现在被认为是基因表达控制的积极参与者。该研究计划的重点是了解核糖开关的结构、生物物理和生化特性，核糖开关是一类广泛存在的感知细胞代谢物的顺式作用调控RNA。核糖开关与公共健康相关，因为它们直接影响细胞的转录、翻译和信使核糖核酸剪接过程。此外，许多核糖开关是新型抗生素的有希望的靶标，因为它们调节人类病原体的基本基因，如金黄色葡萄球菌、炭疽杆菌和霍乱弧菌。我的重点是两种核糖开关，GLMS核酶和甘氨酸核糖开关，它们与目前已知的天然RNA的区别在于它们各自的催化和合作配体结合机制。我提出的这项研究利用了高分辨率结构研究和详细的生化分析的协同作用。本研究的具体目的是：(1)确定GLMS核酶的催化机制；(2)阐明甘氨酸核糖开关与协同配体结合的分子基础；(3)确定甘氨酸核糖开关的高分辨晶体结构。这项研究计划将使我完成我的培训，并为我提供在荧光光谱、核酶动力学和RNA热力学分析方面的新技能。拟议的培训与我的长期目标有关，即建立一个独立的研究计划，致力于了解在生物学中发挥重要催化和调节作用的RNA的结构-功能关系。禤浩焯博士的实验室为这项研究和培训提供了一个特殊的环境，这是因为该实验室过去在催化核糖核酸的结构和生物物理研究方面取得了成功。相关性：所有活着的有机体都必须精确地控制数千个基因的激活或表达。这项研究计划旨在解决基因表达是如何由一组特定的RNA分子控制的，这些RNA分子被认为是有害细菌生存所必需的。由于迫切需要开发新药来对抗对当前抗生素具有耐药性的细菌的崛起，这项工作将为开发抑制这些RNA分子的新型抗生素提供一个起点。

DESCRIPTION (provided by applicant): PROJECT SUMMARY: RNA, once thought of as a passive intermediate in the flow of genetic information, is now recognized as an active participant in the control of gene expression. The focus of this research program is to understand the structural, biophysical, and biochemical properties of riboswitches, a widespread class of cis-acting regulatory RNAs that sense cellular metabolites. Riboswitches are relevant to public health because they directly affect the cellular processes of transcription, translation, and mRNA splicing. Moreover, many riboswitches are promising targets for novel antibiotics because they regulate essential genes in human pathogens such as Staphylococcus aureus, Bacillus anthracis, and Vibrio cholerae. I am focusing on two riboswitches, the glmS ribozyme and the glycine riboswitch, that are distinguished from currently known natural RNAs by their respective catalytic and cooperative ligand binding mechanisms. The research I propose capitalizes on the synergism of high-resolution structural studies and detailed biochemical analyses. The specific aims of this research are: (1) to determine the catalytic mechanism of the glmS ribozyme, (2) to elucidate the molecular basis of cooperative ligand binding by the glycine riboswitch, and (3) to determine a high-resolution crystal structure of the glycine riboswitch. This research program will allow me to complete my training and provide me with new skills in fluorescence spectroscopy, ribozyme kinetics and thermodynamic analyses of RNA. The proposed training is connected to my long-term objective of establishing an independent research program dedicated to understanding the structure-function relationships of RNAs that play important catalytic and regulatory roles in biology. The laboratory of Dr. Adrian Ferre-D'Amare provides an exceptional environment for this research and training, owing to the laboratory's past success in structural and biophysical studies of catalytic RNA. RELEVANCE: All living organisms must precisely control the activation, or expression, of thousands of genes. This research program aims to address how gene expression is controlled by a specific set of RNA molecules that are thought to be necessary for the survival of harmful bacteria. With an urgent need to develop new drugs to combat the rise of bacteria that are resistant to current antibiotics, this work will provide a starting point to develop novel antibiotics that inhibit these RNA molecules.