Structural Biology of RNA

RNA的结构生物学

• 批准号: 6359275
• 负责人: Adrian R. Ferre-D'Amare
• 金额: $ 30.54万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6359275
• 关键词: RNA; X ray crystallography; catalyst; chemical kinetics; chemical structure function; computer simulation; crystallization; enzyme substrate complex; inhibitor /antagonist; intermolecular interaction; lyase; mathematical model; model design /development; molecular dynamics; molecular site; nucleic acid structure; peptide chemical synthesis; physical model; posttranscriptional RNA processing; protein purification; ribonucleoproteins; ribozymes; site directed mutagenesis; structural biology; telomerase; uracil nucleoside

We wish to understand the principles governing the three-dimensional (3D) architecture of biological RNAs and their mechanisms of action. RNAs and proteins are unique among biological macromolecules in being able to self-organize to adopt 3D conformations that are specified by their sequences. It is their 3D structures that enables these macromolecules to carry out the biochemical transformations that underlie all of cell biology. Whereas hundreds of protein structures have been determined at high resolution, the detailed 3D structures of only a handful of biologically-functional RNAs are known. We wish to understand, in atomic detail, how RNAs can fold into compact 3D structures with solvent-inaccessible interiors, how they can catalyze biochemical transformations and how RNA structure is exploited for specific RNA-protein interactions. We study two classes of model systems: catalytic RNAs (ribozymes), and protein enzymes responsible for post-transcriptional RNA modifications. We study the hairpin ribozyme and the Varkud satellite (VS) ribozyme. These two naturally-occurring ribozymes catalyze the same overall chemical transformation, yet appear to have unrelated 3D structures and to use different catalytic mechanisms. We study pseudouridine (psi) synthases, a family of protein enzymes responsible for the most abundant type of post- transcriptional modification of cellular RNAs. These enzymes must modify only specific residues of their substrate RNAs, and have evolved sophisticated means of recognizing the structures of their substrates. Our experimental approach combines X-ray crystallography and biochemistry. We will visualize the ground- state structures of our model macromolecules at atomic or near- atomic resolution by crystallography. The structures will suggest hypotheses about the mechanisms of action of these macromolecules in terms of specific atomic groups and their interactions. These hypotheses will be tested by modifying the candidate atomic groups by either site-directed mutagenesis or synthetic chemistry. The latter is feasible with extant methodology since our model systems are of relatively modest size (less than 50 kDa). Because our model systems are all catalysts, we can then employ the sensitive tools of enzyme kinetics to read out the effects of our targeted perturbations on the activity of the macromolecules. We will also analyze how the structure of our model RNAs changes during the act of catalysis. We will employ the tools of time-resolved crystallography to accomplish this. Finally, we will employ biochemistry and crystallography to analyze how, in eukaryotes, certain nucleolar RNAs scaffold the assembly of psi synthases and accessory proteins into versatile catalytic machines.

我们希望了解生物RNA的三维（3D）结构及其作用机制的原理。 RNA和蛋白质在生物大分子中是独特的，能够自组织以采用由其序列指定的3D构象。 正是它们的3D结构使这些大分子能够进行作为所有细胞生物学基础的生化转化。 尽管已经在高分辨率下确定了数百种蛋白质结构，但只有少数生物功能RNA的详细3D结构是已知的。 我们希望了解，在原子的细节，RNA如何可以折叠成紧凑的三维结构与溶剂无法接近的内部，它们如何可以催化生化转化和RNA结构是如何利用特定的RNA-蛋白质相互作用。 我们研究了两类模型系统：催化RNA（核酶）和负责转录后RNA修饰的蛋白酶。我们研究了发夹状核酶和Varkud卫星核酶。 这两种天然存在的核酶催化相同的整体化学转化，但似乎具有不相关的3D结构，并使用不同的催化机制。 我们研究了假尿苷（psi）脱氢酶，一个负责细胞RNA最丰富的转录后修饰类型的蛋白酶家族。 这些酶必须只修饰其底物RNA的特定残基，并且已经进化出识别其底物结构的复杂方法。 我们的实验方法结合了X射线晶体学和生物化学。 我们将透过结晶学，以原子或近原子的解析度，来显示我们的模型大分子的基态结构. 这些结构将提出关于这些大分子在特定原子团及其相互作用方面的作用机制的假设。 这些假设将通过定点诱变或合成化学修饰候选原子团进行测试。 后者是可行的与现存的方法，因为我们的模型系统是相对温和的大小（小于50 kDa）。 因为我们的模型系统都是催化剂，我们可以使用酶动力学的敏感工具来读出我们的目标扰动对大分子活性的影响。 我们还将分析我们的模型RNA的结构如何在催化过程中发生变化。 我们将采用时间分辨晶体学的工具来实现这一点。 最后，我们将采用生物化学和晶体学来分析，在真核生物中，某些核仁RNA支架组装的psi酶和辅助蛋白到多功能的催化机器。