Crystallographic Studies of Ribosome Functional Sites

描述（由申请人提供）：将开发一种系统，该系统允许在23 S rRNA中插入突变并产生50 S核糖体亚基的高质量晶体。这样的系统将提供在原子分辨率下研究突变的结构效应的手段。例如，将从晶体学上研究rRNA中2482位（海盐蚌）鸟嘌呤至腺嘌呤突变的结构效应。位置2482在肽基转移酶活性位点的结构上是重要的，并且该位点的鸟嘌呤至腺嘌呤突变赋予对氯霉素抗生素的抗性。关于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作用于核糖体的机制。将通过将药物浸泡到H marismortui 50 S核糖体亚基的晶体中来研究evernimicin的结合位点，evernimicin是一种用于治疗多重耐药细菌感染的新实验抗生素。关于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。离子强度和碱基三联体的形成对腺嘌呤的pKa的影响将使用光学熔融和NMR进行研究。由于腺嘌呤在50 S核糖体亚基和其他核酶的活性位点中具有重要的功能，因此更好地理解腺嘌呤的pKa变化的结构背景将有助于理解RNA中的结构-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.