Quality Control in Ribosome Assembly - the Function of Regulatory Proteins

描述(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，核糖体组装中的调控和随后的质量控制，就像翻译的其他步骤一样，是至关重要的。该提案的目的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40S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和定义明确，涉及RNA初级序列的两个变化。然而，它至少需要九个辅助因素，这在酵母中是必不可少的。其中一些因素的活动通常被认为是受调控的，这表明这一步骤对于机械解剖来说足够简单，但也足够复杂，可以对RNA-蛋白质复合体(RNP)组装产生重要的见解。由于核糖体是最保守和最古老的RNPs，这些见解有望具有根本性的重要性。该提案涉及ATPase Fap7的作用，该酶的能量需求功能使其在催化连续的核糖体前重塑步骤中发挥独特的调节作用，推测的内切酶Nob1和解旋酶Rok1。根据目前的数据，我们假设这些步骤中内置的需要能量的构象开关被用来对核糖体组装进行排序和空间和时间调节，以一种响应细胞生长环境变化的方式。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组，发现新的中间体，以及关于关键组装因子在协调空间和时间调节40S组装的构象变化中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关于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点。

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.