Structural Role of IF1 in Translation Initiation

IF1 在翻译起始中的结构作用

• 批准号: 6520502
• 负责人: JOSEPH D PUGLISI
• 金额: $ 25.8万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520502
• 关键词: Escherichia coli; aminoacyl tRNA; aminoglycoside antibiotics; analog; autoradiography; conformation; fluorescence spectrometry; gel mobility shift assay; genetic translation; intermolecular interaction; messenger RNA; microcalorimetry; model design /development; nuclear magnetic resonance spectroscopy; physical model; protein purification; protein structure function; ribosomal proteins; ribosomes; site directed mutagenesis; structural biology; translation factor

DESCRIPTION (provided by applicant): Initiation is a central point in protein synthesis. In prokaryotes, three protein initiation factors participate in assembly of the 70S initiation complex at the correct start codon. All three initiation factors are essential for cell viability, but the precise function for one, IF1, remains unclear. This proposal takes a combined structural and biochemical approach to understanding the role of IF1 in initiation. IF1 has been shown to bind within the A site of the 30S subunit, In the preliminary data, the binding site for IF1 has been localized to the end of a long helix, called the penultimate helix. A model oligonucleotide that corresponds to this helix mimics the ribosomal binding site for IF1 and forms a 1:1 complex suitable for NMR. In specific aim 1, the three-dimensional structure of the IF1 -RNA oligonucleotide complex will be determined by NMR spectroscopy. Biophysical methods, such as titration calorimetry and NMR, will be used to probe the physical origins of specific RNA recognition by IF1. In specific aim 2, the functional effects of IF1 binding to 30S subunits will be probed by measuring tRNA affinities and specificities for the ribosomal P-site using a gel mobility shift assay. Also, IF1 will be fluorescently labeled for binding affinity measurements for 30S and 70S ribosomes. A fluorescent label will also be incorporated into the penultimate helix by mutation to include a BIV Tat peptide binding site, which will bind fluorescent BIV Tat peptide. This will allow ribosomal conformational changes to be monitored. In specific aim 3, the effects on IF1 binding to 30S subunits of antibiotics that disrupt A-site function will be determined. This has important implications for the mechanism of action of these drugs, and for the discovery of novel therapeutic compounds. In the final specific aim (4), the structural and functional homolog of IF1 in eukaryotic organisms will be revealed. The IF1 homolog, which is either eIF2alpha, eIF1A or eIF5A, should bind as well to the eukaryotic 40S subunit A site. Once the homolog is identified, structure determination on protein alone and on the RNA-initiation factor complex will be performed. These studies should reveal how a protein initiation factor specifically recognizes its RNA target, how ribosomal structure is changed by factor binding, and the implications of this binding for ribosome function and the ultimate expression and regulation of genetic information.

描述（由申请人提供）：起始是蛋白质的中心点 合成。在原核生物中，三种蛋白质起始因子参与 在正确的起始密码子处组装 70S 起始复合物。全部三个 起始因子对于细胞活力至关重要，但其精确功能 其中之一，IF1，仍不清楚。该提案结合了结构和 生化方法来了解 IF1 在起始中的作用。 IF1 有 已显示在 30S 亚基的 A 位点内结合，在初步研究中 数据显示，IF1 的结合位点已定位于长螺旋的末端， 称为倒数第二螺旋。与此相对应的模型寡核苷酸 螺旋模拟 IF1 的核糖体结合位点并形成 1:1 复合物 适用于核磁共振。在具体目标1中，IF1的三维结构 -RNA寡核苷酸复合物将通过NMR波谱法测定。 生物物理方法，如滴定量热法和核磁共振，将用于 探究 IF1 特异性 RNA 识别的物理起源。在特定目标下 2、IF1与30S亚基结合的功能效应将通过以下方式探测 使用 a 测量核糖体 P 位点的 tRNA 亲和力和特异性 凝胶迁移率变化测定。此外，IF1 将被荧光标记以进行结合 30S 和 70S 核糖体的亲和力测量。荧光标记也会 通过突变并入倒数第二个螺旋以包含 BIV Tat 肽结合位点，它将结合荧光 BIV Tat 肽。这将 允许监测核糖体构象变化。在具体目标 3 中， 对 IF1 与破坏 A 位点的抗生素 30S 亚基结合的影响 函数将被确定。这对该机制具有重要意义 这些药物的作用，以及发现新的治疗化合物。 在最终的具体目标（4）中，IF1的结构和功能同源物 真核生物将被揭示。 IF1 同源物，它是 eIF2alpha、eIF1A 或 eIF5A 也应与真核 40S 亚基 A 结合 地点。一旦鉴定出同源物，就可以单独确定蛋白质的结构 并对RNA起始因子复合物进行检测。这些研究 应该揭示蛋白质起始因子如何特异性识别其 RNA 目标，核糖体结构如何通过因子结合而改变，以及 这种结合对核糖体功能和最终表达的影响 和遗传信息的调控。