Structural Role of IF1 in Translation Initiation

描述(申请人提供)：起始是蛋白质的中心点 综合。在原核生物中，有三种蛋白质起始因子参与 在正确的起始密码子上组装70年代的起始复合体。三个都是 启动因子是细胞存活所必需的，但其确切的功能 首先，IF1仍不清楚。这项建议采取了结构性和结构性相结合的方式 了解IF1在启动过程中的作用的生化方法。IF1有 已被证明结合在30S亚基的A位点内，在初步 数据，IF1的结合位点已经定位在长螺旋的末端， 称为倒数第二个螺旋。与此相对应的模型寡核苷酸 Helix模拟IF1的核糖体结合部位，形成1：1的复合体 适用于核磁共振。在具体目标1中，IF1的三维结构 -RNA寡核苷酸复合体将通过核磁共振波谱进行测定。 生物物理方法，如滴定量热法和核磁共振，将被用于 探索IF1识别特定RNA的物理来源。以特定的目标 2，探讨IF1与30S亚基结合的功能效应。 测定核糖体P-位点的tRNA亲和力和特异性 凝胶迁移率改变分析。此外，将对IF1进行荧光标记以进行结合 30s和70s核糖体的亲和力测量。一个荧光标签也将 通过突变结合到倒数第二个螺旋中以包括BIV TAT 肽结合部位，可与BIV TAT荧光多肽结合。这将是 允许监测核糖体的构象变化。在具体目标3中， 干扰A-位点的IF1与抗生素30S亚基结合的影响 功能将被确定。这对该机制具有重要的影响。 这些药物的作用以及新的治疗化合物的发现。 在最终的特定目标(4)中，IF1的结构和功能同源 真核生物将会被揭示。IF1同源基因，它是 EIF2α、eIF1a或eIF5A也应该与真核细胞的40S亚基A结合 地点。一旦确定了同源物，就只对蛋白质进行结构确定 并对RNA-起始因子复合体进行了研究。这些研究 应该揭示蛋白质起始因子是如何特异性识别其RNA的 靶点，核糖体结构如何通过因子结合改变，以及 这种结合对核糖体功能和最终表达的影响 以及对遗传信息的监管。

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.