Specificity in Substrate Recognition and Catalysis by RNA Processing Enzymes

项目总结/摘要 基因的表达依赖于许多RNA加工酶的功能，它们的作用是通过调节RNA的合成来实现的。 功能障碍或失调通常与疾病有关。RNA加工的标志 核酸内切酶（如RNA酶E、P、III、Cas9和许多其他核酸内切酶）的作用是作用于大的RNA酶的能力。 细胞中不同的RNA底物的数量，尽管它们中的最佳序列基序有变化， 结合位点。一个关键的例子是核糖核酸酶P（RNase P），这是一种普遍存在的重要RNA加工酶。 在tRNA的5'末端成熟中起主要作用的酶。然而，有充分的证据表明， 细菌核糖核酸酶P有助于调节tRNA、mRNA和其他小RNA;然而，我们缺乏一种 基本了解它如何整合到RNA代谢中。关于这一点，人们所知甚少。 在结构上更复杂的人RNase P酶的特异性和RNA靶点。未来五 多年来，我们的目标是确定E的作用。大肠杆菌RNase P在RNA生物合成中的作用及其调控 使用全转录组分析全面鉴定其RNA底物和切割位点 工具.我们将使用我们在实验室开发的新的高通量生物化学方法来了解如何 与最佳序列基序的差异影响RNase P加工速率。我们将扩展这些研究 研究人类RNase P特异性，并将数据分析与我们对细菌RNase P的研究进行比对。 RNase P.这些结果与来自体内RNase分析的新兴模型的比较 P靶位点将揭示RNase P的内在生物物理性质在多大程度上被改变。 预测其体内功能特异性。体外和体内特异性之间的不连续性 模型将成为更深入研究的目标，因为它们可能代表有趣的偏离 发现新的RNA生物学。与此同时，我们正在确定 RNA酶稳定反应过渡态以完成催化作用。众所周知，在 溶液RNA磷酰基转移反应可以通过逐步或协同机制发生 其进一步相对于过渡态的质子化、成键和电荷分布而变化。的 磷酰基转移机制的内在可塑性提出了酶学的核心问题： 酶的活性位点改变反应过渡态？核糖核酸酶和核酶 催化相同的化学反应，但具有截然不同的活性位点， 过渡国家？我们正在解决这些问题，采用动力学同位素效应（KIE） 分析，以评估提出的机制方案的核糖核酸酶和核酶。的信息 通过帮助改进计算方法，促进 新型催化剂，并揭示了发展基于过渡态的抑制剂的潜力。

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.