Crystallographic Studies of Ribosome Functional Sites

描述(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下研究突变的结构效应的手段。例如，将从结晶学的角度研究鸟嘌呤对rRNA中2482位(Haloarcula Marismortui)腺嘌呤突变的结构影响。在肽基转移酶活性部位，2482位在结构上很重要，该位点鸟嘌呤到腺嘌呤的突变使人对氯霉素产生耐药性。有关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的作用机制。伊维米星是一种用于治疗多重耐药细菌感染的新型实验性抗生素，将通过将该药物浸泡在H marismortui 50s核糖体亚基的晶体中来研究该药物的结合部位。有关该药物结合部位的结构信息可能有助于理解抗生素对核糖体作用的这一新机制。利用光学熔融和核磁共振技术研究离子强度和碱基三元组的形成对腺嘌呤pKA的影响。由于腺嘌呤在50S核糖体亚基和其他核酶的活性部位具有重要的功能，因此更好地理解腺嘌呤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.