RIBOSOME RECYCLING FACTOR(RRF) IN PROKARYOTES

原核生物中的核糖体回收因子（RRF）

• 批准号: 6490215
• 负责人: AKIRA KAJI
• 金额: $ 28.53万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490215
• 关键词: Escherichia coli; RNA binding protein; X ray crystallography; active sites; analog; antibacterial agents; antimetabolites; bacterial proteins; binding sites; crosslink; cryoelectron microscopy; enzyme inhibitors; genetic translation; molecular assembly /self assembly; monoclonal antibody; protein protein interaction; protein structure function; puromycin; ribosomal proteins; ribosomes; suppressor mutations; temperature sensitive mutant; transfer RNA; translation factor; yeast two hybrid system

DESCRIPTION (Applicant's abstract): The long term objective ot tins project is to develop new antibacterial agents targeted against bacterial ribosome recycling factor (RRF) because RRF is an essential factor for prokaryotic translation but not for eukaryotic cytoplasmic protein synthesis. New antibacterial agents are urgently needed because of the emergence of resistant pathogens to the available antibiotics. RRF, together with elongation factor G (EF-G) or release factor 3 (RF3), catalyzes disassembly of the post-termination complex of ribosomes to recycle the ribosomes. In addition, RRF reduces translational error. RRF is a near perfect tRNA mimic. It is postulated that RRF binds to the ribosome and then translocated by EF-G on the ribosome in a fashion similar to tRNA. Inhibitors of tRNA binding to the ribo some and tRNA translocation will be used to examine this hypothesis. The interaction of RRF with ribosomes and EF-G will be studied by electron microscopy. Chemical cross-linking between RRF and the ribosome will be performed. Protection of ribosomal RNA from the effects of chemical agents by bound RRF will be examined to determine the ribosomal binding site of RRF. The possible influence of RRF on the ribosomal binding site of EF-G will also be examined. The gene encoding EF-G or RF3 will be mutagenized to explore the site of interaction with RRF. Other possible biological components which interact with RRF will be determined by identifying the intergenic suppressor genes of temperature sensitive RRF. The yeast two-hybrid system will also be utilized to find additional components that may interact with RRF. The active site of RRF will be explored using monoclonal antibodies against RRF. The mechanism of how RRF reduces translational error will be elucidated. RRF is postulated to release non-cognate or near cognate peptidyl tRNA from the ribosomal P (peptidyl)-site during the chain elongation steps. The release of cognate peptidyl tRNA from the ribosome by RRF and EF-G will be compared with that of the non- or near cognate peptidyl tRNA. At the A (acceptor) site, RRF is postulated to reduce binding of non- or near cognate aminoacyl tRNA by occupying the A site. The effect of RRF on the fidelity of aminoacyl tRNA binding will be tested. Two newly discovered inhibitors of the E. coil RRF reaction, purpuromycin and T. maritima RRF, will be studied as to their mechanism of inhibition by biochemical as well as crystallographic means

描述（申请人的摘要）：长期目标ot tins项目是 开发针对细菌核糖体的新抗菌剂 回收因子（RRF），因为RRF是原核生物的重要因素 翻译，但不适合真核细胞质蛋白合成。新的 由于抗性的出现，迫切需要抗菌剂 可用抗生素的病原体。 RRF，以及伸长因子g （EF-G）或释放因子3（RF3），催化后末端的拆卸 核糖体的复合物以回收核糖体。另外，RRF减少 翻译错误。 RRF是一个接近完美的tRNA模拟物。据推测 RRF与核糖体结合，然后由EF-G易位。 类似于trna的时尚。 tRNA与Ribo的抑制剂和tRNA结合 易位将用于检查这一假设。 RRF与核糖体和EF-G的相互作用将通过电子研究 显微镜。 RRF和核糖体之间的化学交联将是 执行。保护核糖体RNA免受化学剂的影响 将检查绑定的RRF，以确定RRF的核糖体结合位点。这 RRF对EF-G核糖体结合位点的可能影响也将是 检查。编码EF-G或RF3的基因将被诱变以探索该部位 与RRF的相互作用。其他可能相互作用的生物成分 使用RRF将通过识别基因间抑制基因来确定 温度敏感的RRF。酵母双杂交系统也将用于 查找可能与RRF相互作用的其他组件。 RRF的主动部位 将使用针对RRF的单克隆抗体进行探索。 RRF如何减少翻译误差的机制将被阐明。 RRF是 假设是从非同志或接近同源肽基TRNA释放 链条伸长步骤中的核糖体P（肽基）位点。发行 将RRF和EF-G从核糖体中的同源肽基TRNA与 非同源肽基tRNA的非 - 或附近。在A（受体）网站RRF 假设通过 占用A网站。 RRF对氨基酰基tRNA的保真度的影响 绑定将进行测试。 两种新发现的E. coil RRF反应，紫霉素和 T. Maritima RRF将研究其抑制机制 生化和晶体学手段