Biochemical and Genetic Characterization of Ribosome Biogenesis and Functional Diversity

核糖体 是错综复杂的分子机器，它们在翻译 将遗传密码转化为蛋白质。在面包酵母中，核糖体RNA的组装 需要大量的因子和137个核糖体的正确表达 蛋白质编码基因，其中大部分是复制基因。这些事件 导致对前RNA的加工、组装和出口 对脊椎动物和酵母进行了广泛的研究，整个过程非常 很好理解。然而，调控复制基因表达的机制 核糖体蛋白基因及其对核糖体生物发生和功能的影响 目前仍不清楚。最近，我们发现大多数核糖体 蛋白质类似物的表达并不相同，而且在大多数情况下它们是 以正常生长所需的特定比率表达。这一发现认为 反对核糖体蛋白基因的同等和多余的作用。在……里面 这一建议，我们旨在了解调控表达的机制 复制基因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.确定调控机制 复制的核糖体蛋白基因的表达。在这个目标下，我们将 确定复制的基因如何沟通以建立整体表达 核糖体蛋白。我们最近发现了一套相互监管的 复制的核糖体基因，我们将使用它们作为这项研究的模型。这个 启动子、内含子、转录终止与细胞的影响 将监测核糖核酸酶对这些并列基因表达的影响 将确定影响基因间和基因内调控的因素。这个 这项研究的结果不仅将解释核糖体如何表达 蛋白质是配位的，但也将帮助我们理解一种重要的 基因调控一般机制的组成部分。2.了解 复制基因对核糖体生物发生和功能的影响。在这下面 目的，我们将监测改变核糖体蛋白比例对 核糖体生物发生、翻译和细胞功能。复制的基因 将从异源启动子过表达、缺失或突变，并且 对使用表达标签监测的相关蛋白质的影响。其影响 将使用一组标准的表型测试来测量细胞功能， 包括温度敏感性、抗药性和不同碳的使用 消息来源。生长条件对Parparog表达的影响也将 以识别引起核糖体变化的信号 组成和功能。该项目将直接验证这一假设 真核细胞核糖体的功能多样化和 为核糖体生产如何调控提供了一个机械框架 对生长条件的反应。

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.