Quality Control in Ribosome Assembly - the Function of Regulatory Proteins

核糖体组装的质量控制 - 调节蛋白的功能

• 批准号: 8115786
• 负责人: Katrin Karbstein
• 金额: $ 36.87万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-10 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115786
• 关键词: ATP phosphohydrolase; Address; Antibiotics; Binding; Biochemical; Biogenesis; Biological Assay; Boxing; Cells; Complex; Data; Defect; Dissection; Drug Delivery Systems; Environment; Enzymatic Biochemistry; Enzymes; Eukaryota; Event; Failure; Family; Genetic; Genetic Translation; Goals; Individual; Institutes; Investigation; Kinetics; Knowledge; Lead; Link; Literature; Molecular; Molecular Conformation; Nucleotides; Pathway interactions; Performance; Play; Positioning Attribute; Process; Protein Biosynthesis; Proteins; Quality Control; RNA; RNA Processing; RNA Sequences; Regulation; Ribosomal RNA; Ribosomes; Role; Signal Recognition Particle; Site; Spliceosomes; Structure; System; Testing; Time; Translations; Work; Yeasts; base; cancer cell; cell growth; cofactor; endonuclease; fascinate; genetic regulatory protein; helicase; human disease; in vivo; insight; novel; particle; protein complex; public health relevance; rRNA Precursor; reconstitution; research study; tool; yeast genetics

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. PUBLIC HEALTH RELEVANCE: Ribosomes catalyze protein synthesis in all cells. Failure to correctly assembly ribosomes or to correctly regulate assembly can result in serious human disease, suggesting the ribosome assembly pathway as an important drug target. However, knowledge of individual processing steps and their regulation is required and will be garnered in this proposal.

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的人类疾病的基础。因此，与翻译的其他步骤一样，核糖体组装中的调节和随之而来的质量控制至关重要。该提案的目的是揭示有序的组装步骤的基础，这可能构成了质量控制的基础，并在40S核糖体亚基的最终组装步骤中剖析了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的主要序列的两个变化。但是，它至少需要九个附属因素，这在酵母中至关重要。这些因素中的一些活动通常被认为是调节性的，这表明此步骤足以使机械解剖，但要复杂足以产生对RNA-蛋白质复合物（RNP）组装的重要见解。由于核糖体是最保守和古老的RNP，因此预计这些见解至关重要。该提案探讨了ATPase FAP7的作用，ATPase的作用是其能量的函数定位，它在调节作用，假定的核酸内切酶NOB1和HeLicase Rok1中的独特性位置在催化连续的呈核糖体重塑步骤中。基于当前数据，我们假设内置在这些步骤中的能量构象开关用于在空间和时间上调节核糖体组装，以响应细胞生长环境的变化的方式。我们将使用酵母遗传学，生化和生物物理实验和机械酶学的独特组合来解决特定目的：（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。这项工作将导致部分重建促进的核糖体组装，新型中间体的发现以及有关关键组装因子在修复构象变化方面的功能的分子级信息，该构象变化在空间和时间上调节40s组装。此外，了解缓慢，受调节的步骤和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用于药物目的的目标。 公共卫生相关性：核糖体催化所有细胞中的蛋白质合成。无法正确组装核糖体或正确调节装配会导致严重的人类疾病，这表明核糖体组装途径是重要的药物靶标。但是，需要了解个人处理步骤及其法规，并将在此提案中获得。