Structural Biology of RNA

RNA的结构生物学

• 批准号: 6769347
• 负责人: Adrian R. Ferre-D'Amare
• 金额: $ 30.49万
• 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/6769347
• 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.

我们希望了解控制生物 RNA 三维 (3D) 结构的原理及其作用机制。 RNA 和蛋白质在生物大分子中是独一无二的，因为它们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所有细胞生物学基础的生化转化。 尽管已经以高分辨率确定了数百种蛋白质结构，但只有少数具有生物功能的 RNA 的详细 3D 结构是已知的。 我们希望从原子细节上了解 RNA 如何折叠成具有溶剂无法进入的内部的紧凑 3D 结构，它们如何催化生化转化以及如何利用 RNA 结构实现特定的 RNA-蛋白质相互作用。 我们研究两类模型系统：催化 RNA（核酶）和负责转录后 RNA 修饰的蛋白酶。我们研究发夹核酶和 Varkud 卫星 (VS) 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。 我们研究假尿苷 (psi) 合酶，这是一个负责最丰富类型的细胞 RNA 转录后修饰的蛋白质酶家族。 这些酶必须仅修饰其底物 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。 我们将通过晶体学以原子或近原子分辨率可视化模型大分子的基态结构。 这些结构将根据特定原子团及其相互作用提出有关这些大分子作用机制的假设。 这些假设将通过定点诱变或合成化学修饰候选原子团来测试。 后者对于现有的方法是可行的，因为我们的模型系统的大小相对适中（小于 50 kDa）。 因为我们的模型系统都是催化剂，所以我们可以利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。 我们还将分析模型 RNA 的结构在催化过程中如何变化。 我们将利用时间分辨晶体学工具来实现这一目标。 最后，我们将利用生物化学和晶体学来分析，在真核生物中，某些核仁 RNA 如何将 psi 合酶和辅助蛋白组装成多功能催化机器。