RIBOSOME RECYCLING FACTOR(RRF) IN PROKARYOTES

简介(申请人摘要)：TINS项目的长期目标是 开发针对细菌核糖体的新型抗菌剂 循环因子(RRF)，因为RRF是原核生物的必需因子 翻译，但不是真核细胞质蛋白的合成。新的 由于耐药菌株的出现，迫切需要抗菌药物 病原体对可用的抗生素。RRF和延伸率G (EF-G)或释放因子3(RF3)，催化终止后的分解 使核糖体循环的核糖体复合体。此外，RRF还降低了 翻译错误。RRF是一种近乎完美的tRNA模拟物。据推测， RRF与核糖体结合，然后由EF-G在核糖体上转位。 类似于tRNA的时尚。与核糖体和tRNA结合的tRNA抑制剂 易位将被用来检验这一假设。 RRF与核糖体和EF-G的相互作用将用电子研究 显微镜。RRF和核糖体之间的化学交联将是 已执行。保护核糖体RNA免受化学试剂的影响 将检查结合的RRF以确定RRF的核糖体结合位置。这个 RRF对EF-G核糖体结合部位的可能影响也将是 检查过了。编码EF-G或Rf3的基因将被突变以探索该位点 与RRF的互动。其他可能相互作用的生物成分 与RRF的关系将通过鉴定基因间抑制基因来确定 温度敏感型RRF酵母双杂交系统还将用于 查找可能与RRF交互的其他组件。RRF的活性部位 将使用抗RRF的单抗进行探索。 RRF如何减少翻译错误的机制将被阐明。RRF为 推测将非同源或接近同源的多肽tRNA从 核糖体P(肽基)--链延长步骤中的位置。发布了 RRF和EF-G从核糖体中提取的同源多肽tRNA将与 非同源或接近同源的多肽tRNA的同源性。在A(接受者)站点，RRF 假设通过以下方式减少非同源或接近同源的氨基酰基tRNA的结合 占领A地点。RRF对氨基酰tRNA保真度的影响 将测试绑定。 两种新发现的大肠杆菌RRF反应抑制剂，红霉素和 Maritima RRF的抑制作用机制将被研究 生化和结晶学方法

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