Structural Biology of RNA

我们希望了解支配生物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采用由它们的序列指定的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上的生化转化。虽然已经在高分辨率下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。我们希望在原子细节上了解RNA如何折叠成紧凑的3D结构，其内部是溶剂不可访问的，它们如何催化生化转换，以及RNA结构如何被利用来进行特定的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修饰的蛋白质酶。我们研究了发夹状核酶和Varkud卫星核酶。这两种自然产生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。我们研究伪尿苷(Psi)合成酶，这是一个蛋白质酶家族，负责细胞RNA最丰富的转录后修饰类型。这些酶必须只修饰其底物RNA的特定残基，并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射线结晶学和生物化学。我们将通过结晶学在原子或近原子分辨率下可视化我们的模型大分子的基态结构。这些结构将根据特定的原子基团及其相互作用提出关于这些大分子的作用机制的假设。这些假说将通过定点突变或合成化学修饰候选原子基团来检验。后者对于现有的方法学来说是可行的，因为我们的模型系统的规模相对较小(不到50 kDa)。因为我们的模型系统都是催化剂，所以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的结构在催化过程中是如何变化的。我们将使用时间分辨结晶学的工具来实现这一点。最后，我们将使用生物化学和结晶学来分析，在真核生物中，某些核仁RNA是如何将psi合成酶和辅助蛋白组装成多功能催化机器的。

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.