Quality Control in Ribosome Assembly - the Function of Regulatory Proteins

描述（由申请人提供）：核糖体催化所有细胞中的蛋白质合成。因此，成熟和组装的核糖体是许多抗生素的目标。核糖体组装缺陷也是许多严重人类疾病的根源。因此，与其他翻译步骤一样，核糖体组装中的调节和随后的质量控制至关重要。该提案的目标是揭示有序组装步骤的基础，这可能构成质量控制的基础，并剖析酵母 40S 核糖体亚基最终组装步骤中三种调节蛋白的功能。正在研究的步骤相对简单且明确，涉及 RNA 一级序列的两个变化。然而，它至少需要九个辅助因子，这对于酵母来说是必需的。其中一些因子具有通常被认为是调节性的活性，表明这一步骤足够简单，足以进行机械解剖，但又足够复杂，足以深入了解 RNA-蛋白质复合物 (RNP) 的组装。由于核糖体是最保守、最古老的 RNP，因此这些见解预计具有根本重要性。该提案讨论了 Fap7（一种 ATP 酶）、假定的核酸内切酶 Nob1 和解旋酶 Rok1 在催化连续前核糖体重塑步骤中的作用，其能量需求功能使其具有独特的调节作用。根据当前数据，我们假设这些步骤中内置的需要能量的构象开关用于以响应细胞生长环境变化的方式排序并在空间和时间上调节核糖体组装。我们将使用酵母遗传学、生物化学和生物物理实验以及机械酶学的独特组合来实现特定目标：(1) 表征调节 Nob1 的构象变化。 (2)确定Rok1在促进构象变化中的作用。 (3)确定Fap7在核糖体组装中的作用。这项工作将导致促进核糖体组装的部分重建、新型中间体的发现以及关于关键组装因子在协调空间和时间上调节 40S 组装的构象变化中的功能的分子水平信息。此外，对缓慢、受调控步骤的了解以及有关催化这些步骤的蛋白质的信息将使核糖体组装途径成为药物用途的目标。

DESCRIPTION (provided by applicant): Ribosomes catalyze protein synthesis in all cells. Consequently, mature and assembling ribosomes are the target for many antibiotics. Ribosome assembly defects also underlie many serious human diseases. Thus, regulation and ensuing quality control in ribosome assembly, as in other steps of translation, is of key importance. The goal of this proposal is to uncover the basis for ordered assembly steps, which likely form the basis for quality control, and to dissect the function of three regulatory proteins during the final assembly step of the 40S ribosomal subunit in yeast. The step under investigation is relatively simple and well-defined, involving two changes in the primary sequence of the RNA. Yet, it requires at least nine accessory factors, which are essential in yeast. Some of these factors have activities typically considered regulatory, suggesting that this step is simple enough for mechanistic dissection, yet complex enough to yield important insight into RNA-protein complex (RNP) assembly. Since ribosomes are the most conserved and ancient RNPs, these insights are expected to be of fundamental importance. This proposal addresses the role of Fap7, an ATPase, whose energy-requiring function positions it uniquely for a regulatory role, the putative endonuclease Nob1, and the helicase Rok1, in catalyzing successive pre-ribosome remodeling steps. Based on current data we hypothesize that energy-requiring conformational switches built into these steps are used to order and spatially and temporally regulate ribosome assembly, in a manner that is responsive to changes in the cellular growth environment. We will use a unique combination of yeast genetics, biochemical and biophysical experiments, and mechanistic enzymology to address Specific Aims: (1) Characterize a Conformational Change that Regulates Nob1. (2) Determine the Role of Rok1 in Promoting a Conformational Change. (3) Establish the Role of Fap7 In Ribosome Assembly. This work will lead to partial reconstitution of facilitated ribosome assembly, the discovery of novel intermediates and molecular-level information about the function of key assembly factors in orchestrating a conformational change that spatially and temporally regulates 40S assembly. In addition, knowledge of slow, regulated steps and information about the proteins catalyzing these steps will allow the ribosome assembly pathway to be targeted for drug purposes.