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靶标。核糖体组装缺陷也是许多严重人类疾病的基础。因此，在核糖体组装过程中的调节和随后的质量控制，就像在翻译的其他步骤中一样，是至关重要的。该提案的目标是揭示有序组装步骤的基础，这可能形成质量控制的基础，并在酵母中40 S核糖体亚基的最终组装步骤中剖析三种调节蛋白的功能。研究中的步骤相对简单且定义明确，涉及RNA一级序列的两个变化。然而，它需要至少九个辅助因子，这是酵母中必不可少的。这些因子中的一些具有通常被认为是调节性的活性，这表明这一步骤对于机械解剖来说足够简单，但对于RNA-蛋白质复合物（RNP）组装来说足够复杂，从而产生重要的见解。由于核糖体是最保守和最古老的RNP，这些见解预计将具有根本的重要性。该建议解决了Fap 7的作用，一个ATP酶，其能量需要功能的位置，它独特的调节作用，推定的核酸内切酶Nob 1，和解旋酶Rok 1，在催化连续的前核糖体重塑步骤。基于目前的数据，我们假设，需要能量的构象开关内置到这些步骤被用来命令和空间和时间调节核糖体组装，在一种方式，是响应于细胞生长环境的变化。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解决特定目标：（1）表征调节Nob 1的构象变化。(2)确定Rok 1在促进构象变化中的作用。(3)Fap 7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，发现新的中间体和分子水平上的信息的功能的关键组装因子在编排的构象变化，空间和时间调节40 S组装。此外，了解缓慢的、受调控的步骤以及关于催化这些步骤的蛋白质的信息将允许核糖体组装途径被靶向用于药物目的。

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.