RIBOSOME RECYCLING FACTOR(RRF) IN PROKARYOTES

描述（申请人摘要）：本项目的长期目标是 开发针对细菌核糖体的新型抗菌药物 循环因子（RRF），因为RRF是原核生物的必需因子， 翻译，但不用于真核细胞质蛋白质合成。新 由于耐药菌株的出现， 病原体对可用抗生素的反应。RRF，连同延伸因子G （EF-G）或释放因子3（RF 3），催化终止后细胞的解体。 核糖体的复合物来回收核糖体。此外，RRF降低了 翻译错误RRF是一种近乎完美的tRNA模拟物。据推测 RRF结合到核糖体上，然后通过核糖体上的EF-G以一种 类似于tRNA。tRNA与核糖体结合的抑制剂和tRNA 易位将被用来检验这一假设。 RRF与核糖体和EF-G的相互作用将通过电子转移来研究。 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免受化学试剂的影响 将检查结合的RRF以确定RRF的核糖体结合位点。的 RRF对EF-G的核糖体结合位点的可能影响也将是 考察编码EF-G或RF 3的基因将被诱变以探索该位点 与RRF的互动。其他可能的相互作用的生物成分 与RRF将确定通过鉴定基因间抑制基因， 温度敏感RRF。酵母双杂交系统也将用于 查找可能与RRF交互的其他组件。RRF的活性部位 将使用抗RRF的单克隆抗体进行探索。 RRF如何减少平移误差的机制将被阐明。RRF是 假定从细胞中释放非同源或接近同源的肽基tRNA， 核糖体P（肽基）-位点在链延长步骤。释放 通过RRF和EF-G从核糖体中提取的同源肽基tRNA将与 非同源或接近同源的肽基tRNA。在A（受体）位点，RRF 假定通过以下方式减少非同源或接近同源的氨酰tRNA的结合： 占据A区。RRF对氨酰tRNA保真度的影响 将测试绑定。 两个新发现的E.螺旋RRF反应，紫嘌呤霉素和 T. maritima RRF将通过以下方法研究其抑制机制 生物化学和晶体学手段

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