NUCLEIC ACID PROBES OF RIBOSOMAL STRUCTURE AND FUNCTION

核糖体结构和功能的核酸探针

• 批准号: 6386203
• 负责人: BARRY S. COOPERMAN
• 金额: $ 30.05万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386203
• 关键词: Escherichia coli; RNA directed DNA polymerase; SDS polyacrylamide gel electrophoresis; antibiotics; conformation; crosslink; drug resistance; high performance liquid chromatography; matrix assisted laser desorption ionization; nucleic acid probes; nucleic acid sequence; oligonucleotides; photochemistry; photolysis; protein biosynthesis; radiotracer; ribosomal RNA; ribosomes

The ribosome is the unique site of protein biosynthesis in all cells, and as such a detailed understanding of its structure and function is of fundamental importance to the more general understanding of cellular function at the molecular level. Aside from its intrinsic importance to the basic comprehension of life processes, better understanding of ribosomal function could have important therapeutic consequences. Many antibiotics in current clinical use, such as tetracycline, erythromycin and other macrolides, neomycin and other aminoglycosides, and chloramphenicol target ribosomes as their sites of action. Interest in these ribosomal antibiotics has been growing as bacterial resistance to beta-lactams and quinolines has become more widespread. Several drug companies are now devoting considerable resources toward synthesizing analogues and derivatives of ribosomal antibiotics that overcome bacterial resistance. Better understanding of ribosomal structure and function will be especially important for antibiotics, such as macrolides, where resistance is based on changes in ribosome structure. Our studies will be carried out on the E. coli ribosome, which is by far the best characterized by the studies of many groups, including our own. However, given the considerable conservation of ribosome structure throughout evolution the results we obtain should also be useful for understanding ribosomes from other organisms. The overall goal of this proposal is to describe conformational changes that the ribosome undergoes during specific steps of its functional cycle and how mutations and antibiotic binding affect these changes. We propose to do this by forming defined photocrosslinks from rRNA sites within the ribosome that have been targeted on the basis of their importance for ribosome structure and function, taking advantage of the intrinsic ability of the photocrosslinking process to sample all conformations in solution. Such crosslinks will be formed in different functional states, in wild-type and mutant ribosomes, and in the presence and absence of antibiotics. The structural constraints represented by such crosslinks, along with constraints generated by other approaches, will be used to model structures of the ribosome in specific functional states, using crystal structures of 70S ribosomes and 30S and SOS subunits as initial structures. As our major approach we will continue and refine the use of radioactive, photolabile derivatives of oligonucleotides having sequences complementary to rRNA sequences (PHONTs). Such probes bind to their targeted sequences in intact ribosomal subunits, and, on photolysis, incorporate into neighboring ribosomal components that can subsequently be identified. We also will develop a second approach based on site-specific introduction of photolability into intact rRNA (IPHOR - intact photolabile RNA) to obtain similar information for rRNA sites that are either inaccessible to PHONTs or where the use of PHONTs induces major conformational change.

核糖体是所有细胞中蛋白质生物合成的独特部位，因此，详细了解核糖体的结构和功能对于在分子水平上更全面地了解细胞功能具有重要意义。除了对生命过程的基本理解具有内在重要性外，更好地理解核糖体功能可能具有重要的治疗效果。目前临床上使用的许多抗生素，如四环素、红霉素等大环内酯类、新霉素等氨基糖苷类药物，以及氯霉素等都以核糖体为作用靶点。随着细菌对β-内酰胺类和喹啉类抗生素的耐药性变得越来越普遍，人们对这些核糖体抗生素的兴趣也在增长。几家制药公司现在正在投入大量资源来合成核糖体抗生素的类似物和衍生物，以克服细菌耐药性。更好地了解核糖体的结构和功能对抗生素尤其重要，例如大环内酯类药物，其耐药性是基于核糖体结构的变化。我们的研究将在大肠杆菌核糖体上进行，这是目前为止最好的特点，许多小组的研究，包括我们自己。然而，鉴于核糖体结构在整个进化过程中的相当大的保守性，我们所获得的结果也应该有助于理解来自其他生物的核糖体。这一建议的总体目标是描述核糖体在其功能周期的特定步骤中经历的构象变化，以及突变和抗生素结合如何影响这些变化。我们建议通过从核糖体内的rRNA位点形成定义的光交联来实现这一点，这些rRNA位点是根据它们对核糖体结构和功能的重要性而被靶向的，利用光交联过程的内在能力来采样溶液中的所有构象。这种交联会在不同的功能状态下形成，在野生型和突变型核糖体中，以及在抗生素存在和不存在的情况下。这种交联键所代表的结构约束以及其他方法产生的约束将被用来模拟特定功能状态下的核糖体结构，使用70年代核糖体和30年代核糖体和SOS亚基的晶体结构作为初始结构。作为我们的主要方法，我们将继续并改进具有与rRNA序列互补的序列的放射性、耐光性寡核苷酸(PHONTs)的使用。这种探针在完整的核糖体亚基中与其靶序列结合，并在光解时并入相邻的核糖体成分中，这些成分随后可以被识别。我们还将开发第二种方法，基于对完整rRNA(IPHOR完整可光RNA)的位点特异性引入光解性，以获得PHONTs无法访问或PHONTs的使用导致重大构象变化的rRNA位点的类似信息。