The Role of Ribosomal RNA Modifications

描述（由申请人提供）：建议对核糖体RNA （rRNA）的位点特异性修饰进行研究，其中将采用合成，生物物理和生物学方法的结合。这项工作的长期目标是了解修饰的核苷酸在rRNA中的意义及其在调节核糖体功能（即翻译）中的作用。设计新的靶向rRNA的药物作为潜在的抗菌药物将最终依赖于对rRNA结构-功能关系的基本理解。拟建项目的短期目标是获得关于假尿嘧啶及其类似物在调节核糖体构象变化和与蛋白质合成有关的动力学方面的作用的详细信息。将合成代表细菌大亚基RNAs（螺旋69）结构域IV暴露发夹的修饰rna，并利用各种生物物理技术对其进行表征。初步研究揭示了人类和细菌H69 rna的序列和结构差异，文献报道表明H69对正常核糖体功能至关重要，使该核糖体区域成为理想的药物靶向位点。研究计划的具体目标是：1)通过各种生物物理研究（核磁共振、圆二色性、荧光光谱）表征人类和细菌中H69的构象开关机制；2)对全长核糖体rna和核糖体进行化学探测和生物分析，揭示模型系统中观察到的结构变化是否与自然系统相关；3)鉴定和表征对细菌螺旋69比人类变体具有特异性的配体。并能选择性抑制细菌核糖体功能。这些目标结合了多个研究领域（有机合成、RNA修饰、RNA生物物理学和化学探测）的优势，以解决影响人类健康和防御耐药细菌的关键问题。

DESCRIPTION (provided by applicant): A study of site-specific modifications in ribosomal RNA (rRNA) is proposed in which a combination of synthetic, biophysical, and biological approaches will be taken. The long-term goal of the work is to understand the significance of modified nucleotides in rRNA and their roles in regulation of ribosome function, namely translation. The design of novel rRNA-targeting drugs as potential antibacterials will ultimately rely on a fundamental understanding of rRNA structure- function relationships. The short-term goals of the proposed project are to obtain detailed information regarding the roles of pseudouridine and its analogues in regulating ribosome conformation changes and dynamics as they relate to protein synthesis. Modified RNAs representing the exposed hairpin in domain IV of the bacterial large subunit rRNAs (helix 69) will be synthesized and characterized by various biophysical techniques. Preliminary studies revealed sequence and structural differences between human and bacterial H69 RNAs, and literature reports indicate that H69 is essential for normal ribosome function, making this region of the ribosome an ideal drug-targeting site. The specific aims of the research plan are to: 1) characterize the conformational switching mechanism in H69 in humans and bacteria through a variety of biophysical studies (NMR, circular dichroism, fluorescence spectroscopy), 2) use chemical probing and biological assays on full-length ribosomal RNAs and ribosomes to reveal whether structural changes observed in model systems are relevant in natural systems, and 3) identify and characterize ligands that have specificity for bacterial helix 69 over the human variant, and can selectively inhibit bacterial ribosome function. Together these aims combine the strengths of multiple areas of research (organic synthesis, RNA modification, RNA biophysics, and chemical probing) t address key issues that will impact human health and defense against resistant bacteria. PUBLIC HEALTH RELEVANCE: The protein synthesis machinery, the ribosome, is essential to all living organisms, and catalyzes key steps in translating genetic information stored in the form of a nucleic acid to a functionally important protein. This machine depends on precise molecular interactions in order to maintain fidelity and carry out its function essentially without errors. The pseudouridine modification, which is abundant in ribosomal RNA, plays a key role in maintaining such fidelity. One region containing conserved pseudouridine modifications, helix 69, is essential for normal cell growth. The exact roles of pseudouridine in regulating ribosome function are currently unknown. The aims of this proposal are to elucidate the molecular and physical basis for helix 69 dynamics and function, and to determine key differences between human and bacterial ribosome structure and function. This information will be used to design and select for ligands that selectively target bacterial ribosomes, and could lead to the development of potential therapeutics for drug-resistant and/or pathogenic bacteria, thus having an impact on human health.