Quality Control in Ribosome Assembly - the Function of Regulatory Proteins

描述（由申请人提供）：核糖体在所有细胞中催化蛋白质合成。因此，成熟和组装核糖体是许多抗生素的靶标。核糖体组装缺陷也是许多严重人类疾病的基础。因此，调控和随后的核糖体组装质量控制，如在翻译的其他步骤，是至关重要的。本研究的目标是揭示有序组装步骤的基础，这可能是质量控制的基础，并剖析酵母40S核糖体亚基最后组装步骤中三种调节蛋白的功能。所研究的步骤相对简单且定义明确，涉及RNA初级序列的两个变化。然而，它需要至少九种辅助因子，这是酵母必不可少的。其中一些因子具有通常被认为具有调节作用的活性，这表明这一步骤对于机械解剖来说足够简单，但对于rna -蛋白复合物（RNP）组装来说又足够复杂，可以产生重要的见解。由于核糖体是最保守和最古老的RNPs，这些见解被认为是至关重要的。本研究提出了Fap7（一种atp酶，其需要能量的功能使其具有独特的调节作用）、推测的内切酶Nob1和解旋酶Rok1在催化连续的核糖体前重构步骤中的作用。根据目前的数据，我们假设这些步骤中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对核糖体组装进行排序和空间和时间调节。我们将使用酵母遗传学，生化和生物物理实验以及机械酶学的独特组合来解决具体目标：(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，发现新的中间体和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，对缓慢的、受调节的步骤的了解以及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目的的靶标。

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.