Crystallographic Studies of Ribosome Functional Sites

描述（由申请人提供）：将开发一种系统，允许在23S rRNA中插入突变，并产生高质量的50S核糖体亚基晶体。这样的系统将提供在原子分辨率上研究突变的结构效应的手段。例如，鸟嘌呤对rRNA中2482位（Haloarcula marismortui）腺嘌呤突变的结构影响将被晶体学研究。2482位在肽基转移酶活性位点上具有重要的结构意义，该位点的鸟嘌呤到腺嘌呤突变使其对氯霉素类抗生素产生耐药性。G2482突变的结构信息将有助于理解肽基转移酶反应和抗生素对核糖体作用的机制。evernimicin是一种用于治疗多重耐药细菌感染的新型实验性抗生素，将通过将药物浸泡在hmarismortui 50S核糖体亚基晶体中来研究其结合位点。这种药物结合位点的结构信息可能有助于理解这种抗生素作用于核糖体的新机制。离子强度和碱基三元组的形成对腺嘌呤pKa的影响将通过光学熔融和核磁共振进行研究。由于腺嘌呤在50S核糖体亚基和其他核酶的活性位点具有重要的功能，因此更好地了解腺嘌呤pKa变化的结构背景将有助于理解RNA的结构-功能关系。

DESCRIPTION (provided by applicant): A system will be developed that allows insertion of mutations in the 23S rRNA and produces high quality crystals of the 50S ribosomal subunit. Such a system will provide the means to study the structural effects of mutations at atomic resolution. For example, the structural effects of guanine to adenine mutations at position 2482 (Haloarcula marismortui) in rRNA will be studied crystallographically. Position 2482 is structurally important at the peptidyl transferase active site, and guanine to adenine mutations at this site confers resistance to chloramphenicol antibiotic. Structural information about mutations at G2482 will contribute to understanding the mechanisms of the peptidyl transferase reaction and antibiotic action on ribosomes. The binding site of evernimicin, a new experimental antibiotic used to treat multidrug-resistant bacterial infections, will be studied by soaking the drug into crystals of H marismortui 50S ribosomal subunits. Structural information about the binding site of this drug may contribute to understanding this novel mechanism of antibiotic action on ribosomes. The effects of ionic strength and formation of base triples on the pKa of adenines will be studied using optical melting and NMR. Because adenines are functionally important in the active site of the 50S ribosomal subunit and other ribozymes, a better understanding of the structural contexts in which the pKa of adenines changes will contribute to understanding structural-function relationships in RNA.