Biochemical and Genetic Characterization of Ribosome Biogenesis and Functional Diversity

*核糖体*是复杂的分子机器，在将遗传密码翻译成蛋白质的过程中发挥着核心作用。在面包酵母中，组装核糖体RNA需要大量的因子和137个核糖体蛋白编码基因的正确表达，其中大部分是重复基因。导致前rRNA加工、组装和出口的事件在脊椎动物和酵母中得到了广泛的研究，整个过程也非常清楚。然而，调控复制的核糖体蛋白基因表达的机制及其对核糖体生物发生和功能的影响仍不清楚。最近，我们发现，大多数核糖体蛋白并不是同等表达的，而且在大多数情况下，它们是以正常生长所需的特定比例表达的。这一发现反对核糖体蛋白基因的等同和多余的作用。在这项建议中，我们旨在了解复制基因表达的调控机制及其对核糖体生产和功能的影响。因此，我们提出了两个具体目标：1.确定复制核糖体蛋白基因表达的调控机制。在这个目标下，我们将确定复制的基因如何交流，以确定核糖体蛋白的整体表达。我们最近发现了一组相互调控的复制核糖体基因，我们将把它们作为这项研究的模型。将监测启动子、内含子、转录终止和细胞核糖核酸酶对这些准基因表达的影响，并确定影响基因间和基因内调控的因素。这项研究的结果不仅将解释核糖体蛋白的表达是如何协调的，而且还将帮助我们理解基因调控一般机制的一个重要组成部分。2.了解复制基因对核糖体生物发生和功能的影响。在这一目标下，我们将监测改变核糖体蛋白质比例对核糖体生物发生、翻译和细胞功能的影响。复制的基因*将从异源启动子中过度表达、缺失或突变，并使用表达标签监测对相关蛋白质的影响。对细胞功能的影响将使用一套标准的表型测试来衡量，包括温度敏感性、耐药性和不同碳源的使用。为了识别引起核糖体组成和功能变化的信号，还将分析生长条件对Paralog表达的影响。该项目将直接验证真核生物核糖体功能多样化的假说，并为核糖体生产如何对生长条件作出反应提供一个机械框架。

* **Ribosomes*are intricate molecular machines that play a central role in translating the*genetic code into proteins. In baker's yeast, assembly of the ribosomal RNA*requires a large number of factors and the correct expression of 137 ribosomal*protein-coding genes the majority of which are duplicated genes. The events*leading to the processing of pre-rRNAs, assembly and export have been*extensively studied in vertebrates and yeast, and the overall process is very*well understood. However, the mechanism regulating the expression of duplicated*ribosomal protein genes and its impact on ribosome biogenesis and function*remain unclear. Recently, we discovered that the majority of the ribosomal*protein paralogs are not expressed equally and that in most cases they are*expressed in a specific ratio required for normal growth. This finding argues*against an equal and redundant role for the ribosomal protein genes paralogs. In*this proposal, we aim at understanding the mechanism regulating the expression*of duplicated genes and its impact on ribosome production and function. Accordingly,*we propose two specific aims: 1. Determine the mechanism regulating the*expression of duplicated ribosomal protein genes. Under this aim, we will*determine how duplicated genes communicate to establish the overall expression*of ribosomal proteins. We have recently identified a set of inter-regulated*duplicated ribosomal genes, and we will use them as a model for this study. The*impact of promoters, intron, transcription termination and cellular*ribonucleases on the expression of these paralogs will be monitored and*elements that affect inter and intragene regulation will be identified. The*outcome of this study will not only explain how the expression of ribosomal*proteins is coordinated but will also help us to understand an important*component of the general mechanism of gene regulation. 2. Understand the*impact of duplicated genes on ribosome biogenesis and function. Under this*aim, we will monitor the impact of changing the ratio of ribosomal proteins on*ribosome biogenesis, translation and cellular functions. The duplicated genes*will be overexpressed from a heterologous promoter, deleted or mutated and the*impact on the associated proteins monitored using expression tags. The impact*on cell function will be measured using a standard set of phenotypic tests,*including temperature sensitivity, drug resistance and use of different carbon*sources. The effects of growth conditions on the paralogs expression will also*be analyzed in order to identify signals inducing changes in the ribosome*composition and function. This project will directly verify the hypothesis*of paralogs driven functional diversification of eukaryotic ribosomes and*provides a mechanistic frame for how ribosome production is modulated in*response to growth conditions.**