Function and Mechanism of RNA Modification

核糖体是合成所有蛋白质的迷人的分子机器。因此，核糖体是纳米装配机的一个主要例子，这是一种能够在纳米尺度上构建广泛功能部件的机器。核糖体由两个亚基组成，每个亚基都含有复杂折叠的大RNA(核糖核酸)和大量相互交织的蛋白质，这就提出了所有这些成分是如何结合在一起的问题。我的研究计划的长期目标是将核糖体生物发生理解为组装复杂的RNA-蛋白质机器的范例。核糖体的形成始于核糖体RNA(RRNA)的转录、修饰和折叠。在过去的几年里，我一直专注于tRNA中假尿苷形成的分子机制，作为一个简单的RNA修饰的模型系统。我现在提议将这些研究扩展到其他tRNA修饰和rRNA修饰。使用细菌模型系统，我们将特别分析RNA转录、修饰和折叠的相互作用，因为这些过程同时发生，并可能在核糖体组装的早期阶段相互影响。我们综合运用生化、生物物理和分子生物学技术，提出了三个主要问题：1.假性尿苷和其他修饰是如何引入转移RNA的？继续我们之前的研究，我将评估伪尿苷合成酶如何与tRNA结合，并在局部揭开这个小的模型RNA。我们还将通过分析潜在的中间体来解决假尿嘧啶催化的缓慢性质，这是确定这些酶的催化机制的重要一步。最后，我们将剖析tRNA甲基化的动力学及其对RNA折叠的影响，以及作为最常见的RNA修饰的甲基化和假尿嘧啶的相互作用。RNA修饰酶对细菌细胞有什么重要的生物学功能？在这里，我问为什么细菌细胞有许多RNA修饰酶，如果大多数不是必需的，我认为它们的好处在压力条件下将是最明显的。如果是这样的话，在应激条件下，RNA修饰如何影响核糖体的形成和功能？因此，我们还将为后续的体外研究确定最关键的rRNA修饰酶。此外，我们还验证了RNA修饰酶作为RNA伴侣促进RNA折叠的假说。在23S rRNA中，伪尿苷是如何形成的？它们对RNA折叠和核糖体组装有何作用？我将验证修饰酶识别23S rRNA结构的假说。建立共转录的23S rRNA修饰系统将是迈向体外大亚基组装系统的第一步，允许在分析RNA修饰和折叠的同时系统地添加核糖体蛋白质。我提出的研究将提供重要的见解(1)修饰对RNA折叠的影响，(2)不同RNA修饰的时间和相互作用，(3)RNA修饰的细胞功能，以及(4)RNA折叠和修饰对转录的依赖。我们对参与翻译的短和长细菌RNA的研究将产生很可能对所有生物体都适用的知识，因为这些过程是高度保守的。最终，我的研究将导致建立一个实验系统，利用复杂的生物化学实验研究大核糖体亚单位的体外组装。这也将允许操纵核糖体组装，目标是构建新的基于核糖体的纳米组装体，使新化合物的有效合成成为可能。

Ribosomes are fascinating molecular machines that synthesize all proteins. As such, ribosomes are a prime example of a nanoassembler, a machine capable of constructing a wide range of functional parts on the nanometer scale. The ribosome consists of two subunits each containing large, intricately folded RNAs (ribonucleic acids) and numerous interwoven proteins, raising the question of how all these components come together. It is the long-term goal of my research program to understand ribosome biogenesis as a paradigm for the assembly of complex RNA-protein machines. Ribosome formation begins with the transcription, modification and folding of ribosomal RNA (rRNA). In the past years, I have focused on the molecular mechanism of pseudouridine formation in tRNA as a simple model system for RNA modification. I am now proposing to extend these studies to other tRNA modifications and to rRNA modifications. Using a bacterial model system, we will in particular analyze the interplay of RNA transcription, modification and folding as these processes occur simultaneously and may influence each other during the early stages of ribosome assembly. We apply a combination of biochemical, biophysical and molecular biology techniques asking three main questions:1. How are pseudouridines and other modifications introduced into transfer RNA? Continuing our previous research, I will assess how pseudouridine synthases bind tRNA and locally unfold this small model RNA. We will also address the slow nature of pseudouridylation catalysis by analyzing potential intermediates, an important step towards identifying the catalytic mechanism of these enzymes. Lastly, we will dissect the kinetics of tRNA methylation and the impact thereof on RNA folding as well as the interplay of methylation and pseudouridylation as the most common RNA modifications.2. What is the biologically important function of RNA modification enzymes for the bacterial cell? Here, I ask why the bacterial cell has many RNA modification enzymes if most are not essential, and I suggest that their benefit will be most apparent under stress conditions. If so, how do RNA modifications impact ribosome formation and function under stress conditions? Thereby, we will also identify the most critical rRNA modification enzymes for subsequent in vitro studies. Additionally, we test the hypothesis that RNA modification enzymes act as RNA chaperones contributing to RNA folding.3. How are pseudouridines formed site-specifically in 23S rRNA and how do they contribute to RNA folding and ribosome assembly?I will test the hypothesis that modification enzymes recognize 23S rRNA structure that is folded co-transcriptionally. The establishment of a co-transcriptional 23S rRNA modification system will be the first step towards an in vitro large subunit assembly system, allowing the systematic addition of ribosomal proteins while analyzing RNA modification and folding.My proposed research will provide significant insight (1) into the impact of modification on RNA folding, (2) into the timing and interplay of different RNA modifications, (3) into the cellular function of RNA modification, and (4) into the dependence of RNA folding and modification on transcription. Our studies of short and long bacterial RNAs involved in translation will generate knowledge that will very likely hold true for all organisms as these processes are highly conserved. Ultimately, my research will lead to an experimental system for studying assembly of the large ribosomal subunit in vitro using sophisticated biochemistry experiments. This will also allow manipulation of ribosome assembly with the goal of constructing novel ribosome-based nanoassemblers that will enable the efficient synthesis of novel compounds.