The Role of Ribosomal RNA Modifications

描述(申请人提供)：提出了一项关于核糖体RNA(RRNA)位点特异性修饰的研究，其中将采用合成、生物物理和生物学方法相结合的方法。这项工作的长期目标是了解rRNA中修饰核苷酸的意义以及它们在核糖体功能调节中的作用，即翻译。作为潜在抗菌药物的新型rRNA靶向药物的设计最终将依赖于对rRNA结构-功能关系的基本了解。拟议项目的短期目标是获得关于假尿苷及其类似物在调节核糖体构象变化和动力学方面的作用的详细信息，因为它们与蛋白质合成有关。将合成代表细菌大亚基rRNA(螺旋69)结构域IV中暴露的发夹的修饰RNA，并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是正常核糖体功能所必需的，使该核糖体区域成为理想的药物靶点。该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、圆二色谱、荧光光谱)表征H69在人类和细菌中的构象转换机制；2)对全长核糖体RNA和核糖体使用化学探测和生物分析来揭示在模型系统中观察到的结构变化是否与自然系统相关；以及3)识别和表征对细菌螺旋69具有相对于人类变体的特异性并且能够选择性地抑制细菌核糖体功能的配体。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，以解决将影响人类健康和防御耐药细菌的关键问题。 与公共健康相关：蛋白质合成机制，即核糖体，对所有活着的有机体都是必不可少的，并催化将以核酸形式存储的遗传信息转化为具有重要功能的蛋白质的关键步骤。这台机器依靠精确的分子相互作用来保持保真度，并基本上没有错误地执行其功能。伪尿苷修饰在核糖体RNA中含量丰富，在保持这种保真度方面发挥了关键作用。一个含有保守的假尿苷修饰的区域，螺旋69，对于正常的细胞生长是必不可少的。假性尿苷在核糖体功能调节中的确切作用目前尚不清楚。这项建议的目的是阐明螺旋69动力学和功能的分子和物理基础，并确定人类和细菌核糖体结构和功能之间的关键差异。这些信息将被用于设计和选择选择性靶向细菌核糖体的配体，并可能导致开发抗药性和/或病原菌的潜在疗法，从而对人类健康产生影响。

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.