Catalysis and Cooperativity in Regulatory RNAs

项目概述：RNA，曾经被认为是遗传信息流动中的被动中间体，现在被认为是基因表达控制的积极参与者。该研究项目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顺式作用调节rna，可感知细胞代谢物。核糖开关与公众健康相关，因为它们直接影响转录、翻译和mRNA剪接的细胞过程。此外，许多核糖开关是新型抗生素的有希望的靶点，因为它们调节人类病原体（如金黄色葡萄球菌、炭疽芽孢杆菌和霍乱弧菌）的必需基因。我主要关注两种核开关，glmS核酶和甘氨酸核开关，它们与目前已知的天然rna不同，它们具有各自的催化和配合配体结合机制。我提出的研究利用了高分辨率结构研究和详细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)阐明甘氨酸核开关配合配体结合的分子基础；(3)确定甘氨酸核开关的高分辨率晶体结构。这个研究项目将使我能够完成我的培训，并为我提供荧光光谱，核酶动力学和RNA热力学分析方面的新技能。我的长期目标是建立一个独立的研究项目，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。Adrian Ferre-D'Amare博士的实验室为这项研究和培训提供了一个特殊的环境，因为该实验室过去在催化RNA的结构和生物物理研究方面取得了成功。相关性：所有生物体都必须精确控制成千上万个基因的激活或表达。这个研究项目旨在解决基因表达是如何被一组特定的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现有抗生素具有耐药性的细菌的增加，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.