CHARACTERIZATION OF THE STRUCTURE AND DYNAMICS OF 16S RRNA A-SITE MUTANTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 16S核糖体A位直接参与维持核糖体翻译信使核糖核酸的保真度。A位也是新胺类氨基糖苷类抗生素的靶标。这些抗生素与A位点的结合降低了翻译的保真度，导致非功能性蛋白质产物，导致细胞死亡。最近的X射线结晶学证据表明，核苷酸A1492和A1493在A位内环中的位置和接触在维持mRNA翻译的保真度方面起着重要作用。已有研究表明，抗生素与A位内环的结合会扭曲其结构。与A1492和A1493相反的1408位的同一性在很大程度上控制了真核细胞G1408和原核生物A1408之间的抗生素结合特异性，这是这些抗生素在临床上治疗细菌感染成功的主要原因。近年来，一些细菌菌株已经形成了对新胺类抗生素的耐药性机制。一种机制涉及A位靶点序列的突变。我们的假设是，16S核糖体A位点的突变必须保持一个特征的静电表面，以便在翻译过程中tRNA的反密码子环对mRNA密码子进行同源识别。为了验证这一假设，我们正在进行核磁共振研究，以解决原核和真核野生型A-位点的溶液状态结构，以及菲尔·坎宁安博士的实验室使用他的即时进化方法产生的几个细菌功能突变。本工作重点研究了A1492和A1493位置的A-位构象和动力学的序列依赖性。马克·斯佩勒博士的实验室合成的一种新型抗生素与选定结构的结合研究和结构测定也在进行中，以探索药物设计的新途径。将这些结构与当前公布的数据(核磁共振、X射线、足迹等)进行比较。将揭示药物结合的结构和机制方面，以及提供抗生素耐药性和突变功能的合理化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The 16S ribosomal A-site is directly involved in maintaining the fidelity of mRNA translation by the ribosome. The A-site is also the target of neamine-class aminoglycoside antibiotics. Binding of these antibiotics to the A-site reduces the level of fidelity in translation, resulting in non-functional protein products, resulting in cell death. Recent X-ray crystallographic evidence suggests the positions and contacts of nucleotides A1492 and A1493, within the A-site internal loop, play an important role in maintaining fidelity of mRNA translation. It has been shown that antibiotic binding to the A-site internal loop can distort its structure. The identity of position 1408, opposite to A1492 and A1493 within the A-site internal loop, largely controls the antibiotic binding specificity between Eukaryotes G1408 and Prokaryotes A1408, which is the main rationale behind the clinical success of these antibiotics in treating bacterial infections. In recent years a number of bacterial strains have developed resistance mechanisms to the neamine-class of antibiotics. One mechanism involves mutations in the sequence of the A-site target. Our hypothesis is that mutations of the 16S Ribosomal A-site must maintain a characteristic electrostatic surface for cognate recognition of the mRNA codon by the anti-codon loop of tRNA during translation. To test this hypothesis we are performing NMR studies to solve the solution-state structure for the prokaryotic and eukaryotic wild type A-site, along with several bacterial functional mutants generated in the lab of Dr. Phil Cunningham using his instant evolution methods. This work focuses on the sequence dependence of the A-site conformation and dynamics of positions A1492 and A1493. Binding studies and structure determination of a novel antibiotic, synthesized in the lab of Dr. Mark Spaller, bound to selected constructs are also being performed to explore new avenues in drug design. Comparing these structures to currently published data (NMR, x-ray, footprinting, etc.) will reveal structural and mechanistic aspects of drug binding, as well as provide rationalization of antibiotic resistance and mutant function.