Specificity in Substrate Recognition and Catalysis by RNA Processing Enzymes

项目摘要/摘要 基因的表达依赖于许多RNA加工酶的功能，以及它们的 功能障碍或调节不当往往与疾病有关。RNA加工的一个标志 核酸内切酶(如核糖核酸酶E、P、III、Cas9和许多其他核酸酶)是一种作用于 细胞中不同的RNA底物的数量，尽管它们的最优序列基序存在差异 结合部位。一个关键的例子是核糖核酸酶P(RNaseP)，这是一种普遍存在的必要的RNA加工 在tRNAs的5‘端成熟中起主要作用的酶。然而，有充分的证据表明， 细菌RNase P有助于调节tRNAs、mRNAs和其他小RNA；然而，我们缺乏一种 基本了解它是如何整合到RNA新陈代谢中的。更不知道的是关于 更复杂的人核糖核酸酶P酶的特异性和RNA靶标。在接下来的五年里 几年来，我们的目标是通过以下方式确定大肠杆菌RNaseP在RNA生物合成和调控中的作用 利用转录组分析全面鉴定其RNA底物和裂解位点 工具。我们将使用我们实验室开发的新的高通量生化方法来了解如何 最佳序列基序的变异会影响RNaseP的处理速度。我们将扩大这些研究的范围 研究人类核糖核酸酶P的特异性，并将数据分析与我们对细菌的研究相一致 核糖核酸酶P.这些结果与从体内核糖核酸酶分析得出的新兴模型的比较 P靶点将揭示RNaseP的内在生物物理性质在多大程度上 在体内预测其功能特异性。体外和体内特异性之间的不连续性 模特将成为更深入调查的目标，因为它们可能代表着有趣的背离 发现新的RNA生物学的要点。同时，我们正在确定活跃的站点如何 核糖核酸酶稳定反应过渡态以完成催化作用。这是公认的在 溶液RNA磷酸化转移反应可以通过分步或协同机制进行 这与质子化、成键和过渡态的电荷分布有关。这个 磷酸基转移机制的内在可塑性引发了酶学的核心问题：如何 酶的活性部位会改变反应过渡态，而核糖核酸酶和核酶会改变反应过渡态吗？ 催化相同的化学反应，但具有截然不同的活性中心，稳定相同 过渡状态？我们正在利用动力学同位素效应(KIE)来解决这些问题 对核糖核酸酶和核酶机制方案进行评估的分析。这些信息 获得的成果将产生广泛的影响，帮助改进计算方法，促进 新型催化剂，并揭示了基于过渡态的缓蚀剂的开发潜力。

PROJECT SUMMARY/ABSTRACT Gene expression depends on the function of numerous RNA processing enzymes, and their dysfunction or mis-regulation is often associated with disease. A hallmark of RNA processing endonucleases (such as RNase E, P, III, Cas9 and a host of others) is the ability to act on a large number of different RNA substrates in the cell despite variation from optimal sequence motifs in their binding sites. A key example is ribonuclease P (RNase P), a ubiquitous and essential RNA processing enzyme with a primary role in 5' end maturation of tRNAs. However, there is ample evidence that bacterial RNase P contributes to regulation of tRNAs, mRNAs, and other small RNAs; yet, we lack a basic understanding of how it is integrated into RNA metabolism. Even less is known regarding the specificity and RNA targets of the more structurally complex human RNase P enzyme. In the next five years we aim to define the roles of E. coli RNase P in RNA biosynthesis and regulation by comprehensively identifying its RNA substrates and cleavage sites using transcriptome-wide analysis tools. We will use new high throughput biochemical methods we developed in our lab to learn how variation from optimal sequence motifs affects RNase P processing rates. We will extend these studies to investigate human RNase P specificity and align the data analysis with our studies of bacterial RNase P. Comparison of these results with the emerging model derived from analysis of in vivo RNase P target sites will reveal the extent to which the intrinsic biophysical properties of RNase P are predictive of its functional specificity in vivo. Discontinuities between the in vitro and in vivo specificity models will be targeted for deeper investigation since they are likely to represent interesting departure points for discovering novel RNA biology. In parallel, we are determining how the active sites of RNases stabilize reaction transition states in order to accomplish catalysis. It is well-established that in solution RNA phosphoryl transfer reactions can occur either by step-wise or concerted mechanisms that further vary with respect to protonation, bonding, and charge distribution of the transition state. The intrinsic plasticity of phosphoryl transfer mechanisms raises questions central to enzymology: how do the active sites of enzymes alter reaction transition states?; and, do RNases and ribozymes, that catalyze the same chemical reaction, but with profoundly different active sites, stabilize the same transition states? We are addressing these questions by employing kinetic isotope effect (KIE) analyses to evaluate proposed mechanistic scenarios for RNases and ribozymes. The information gained will have broad impact by helping improve computational methods, facilitating the design of novel catalysts, and revealing the potential for development of transition state based inhibitors.