Structural Biology of RNA

我们希望了解控制生物rna的三维（3D）结构及其作用机制的原则。rna和蛋白质在生物大分子中是独一无二的，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生化转化，这是所有细胞生物学的基础。尽管数以百计的蛋白质结构已经以高分辨率确定，但只有少数具有生物功能的rna的详细3D结构是已知的。我们希望从原子的细节上了解RNA如何折叠成紧凑的3D结构，其内部是溶剂无法进入的，它们如何催化生化转化，以及RNA结构如何被用于特定的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。我们研究假尿嘧啶（psi）合成酶，这是一个负责细胞rna转录后修饰的最丰富类型的蛋白酶家族。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。这些结构将提出关于这些大分子在特定原子群及其相互作用方面的作用机制的假设。这些假设将通过位点定向诱变或合成化学修饰候选原子群来验证。后者在现有方法下是可行的，因为我们的模型系统相对较小（小于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晶体学的工具来完成这项工作。最后，我们将运用生物化学和晶体学来分析真核生物中某些核仁rna是如何支撑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.