Structural Role of IF1 in Translation Initiation

描述（由申请方提供）：起始是蛋白质中的中心点 合成.在原核生物中，三种蛋白质起始因子参与了 在正确的起始密码子处组装70 S起始复合物。所有三 起始因子对于细胞活力是必不可少的，但是精确的功能 其中，IF 1仍不清楚。该提案采取了结构和 生物化学方法来理解IF 1在启动中的作用。IF 1有 已显示结合在30 S亚基的A位点内， 数据显示，IF 1的结合位点位于长螺旋的末端， 叫做倒数第二螺旋。与此对应的模型寡核苷酸 螺旋模拟IF 1的核糖体结合位点并形成1：1复合物 适用于NMR。在具体目标1中，IF 1的三维结构 -RNA寡核苷酸复合物将通过NMR光谱测定。 生物物理方法，如滴定量热法和核磁共振，将用于 探索IF 1识别特定RNA的物理起源。具体目标 2、IF 1与30 S亚基结合的功能效应将通过 使用一种用于测量核糖体P位点的tRNA亲和力和特异性的方法， 凝胶迁移率变动分析。此外，IF 1将被荧光标记用于结合 30 S和70 S核糖体的亲和力测量。荧光标记还将 通过突变并入倒数第二个螺旋以包括BIV达特 肽结合位点，其将结合荧光BIV达特肽。这将 允许核糖体构象变化被监测。在具体目标3中， 对IF 1与破坏A位点的抗生素30 S亚基结合的影响 功能将被确定。这对该机制具有重要意义 这些药物的作用，并发现新的治疗化合物。 在最后的具体目标（4）中， 真核生物将被揭示。IF 1同源物，即 eIF 2 α、eIF 1A或eIF 5A也应该与真核40 S亚基A结合 绝佳的价钱一旦同源物被鉴定， 和RNA-起始因子复合物。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，核糖体结构如何被因子结合改变，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。

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.