NUCLEIC ACID PROBES OF RIBOSOME STRUCTURE AND FUNCTION

核糖体结构和功能的核酸探针

• 批准号: 2192428
• 负责人: BARRY S. COOPERMAN
• 金额: $ 23.11万
• 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 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2192428
• 关键词: conformation; high performance liquid chromatography; nucleic acid probes; nucleic acid sequence; oligonucleotides; photochemistry; protein biosynthesis; 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. Our studies will be carried out ont he 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. We will continue our use of radioactive, photolabile derivatives of oligo DNAs having sequence complementary to single-stranded rRNA sequences for this purpose. Such probes can bind to their targeted sequences in intact ribosomal subunits, and, on photolysis, incorporate into neighboring ribosomal components that can subsequently be identified by methods perfected in our laboratory. We will expand the scope of this work by: varying the size of the spacer linking the photolabile group to the complementary base; varying the site of attachment of the photolabile group within the oligoDNA probe; and using photolabile oligoDNA probes to monitor conformational change in the vicinity of the target rRNA sequence. We also will explore the utilization of a second approach based on the replacement of phosphate with thiophosphate or U with 4-thioU at specific locations within rRNA. The electrophilic sulfurs thus introduced into RNA provide specific sites for the incorporation of photolability (of course, the 4-thioU is itself photolabile). Reconstitution of ribosomes with such modified rRNA, followed by photolysis, will allow neighboring components to be identified for rRNA sites that are inaccessible to oligoDNA probes. The proposed studies will provide information critical for the construction of the three-dimensional structure of the ribosome, a requirement for understanding ribosomal function. 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 s 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.

核糖体是所有细胞中蛋白质生物合成的唯一场所， 因此，对它的结构和功能的详细了解是 对于更广泛地理解细胞的基本重要性 在分子水平上发挥作用。 我们的研究将在E. 大肠杆菌核糖体，这是迄今为止最好的特点，研究 很多人，包括我们自己。 然而，考虑到 核糖体结构在整个进化过程中的保守性，结果我们 获得的结果也有助于理解来自其他核糖体的核糖体。 有机体 我们将继续使用放射性的，不稳定的 具有与单链互补序列的寡DNA衍生物 rRNA序列用于此目的。 这样的探针可以结合到它们的靶向 完整的核糖体亚基中的序列，并且在光解时， 与邻近的核糖体成分结合， 通过我们实验室完善的方法。 我们将扩大这一范围 工作方式：改变连接光不稳定基团的间隔基的大小， 互补碱基;改变光不稳定性的附着位点 组内的寡聚DNA探针;和使用光不稳定寡聚DNA探针， 监测靶rRNA序列附近的构象变化。 我们还将探索第二种方法的利用， 用硫代磷酸盐取代磷酸盐或用4-硫代U取代U， rRNA内的位置。 如此引入RNA的亲电硫 提供用于结合光适应性的特定位点（当然， 4-硫代U本身对光不稳定）。 用这样的核糖体重建核糖体 经过修饰的rRNA，然后进行光解，将允许邻近的组分 鉴定寡聚DNA探针无法接近的rRNA位点。 拟议的研究将提供建设的关键信息 核糖体的三维结构， 了解核糖体的功能。 除了其内在的重要性， 对生命过程的基本理解，更好地理解 核糖体功能可能具有重要的治疗效果。 许多 目前临床使用的抗生素，如四环素、红霉素和 其他大环内酯类、新霉素和其他氨基糖苷类以及氯霉素 靶向核糖体作为其作用位点。 对这些核糖体的兴趣 抗生素的细菌对β-内酰胺类抗生素的耐药性越来越强， 喹啉已经变得更加普遍。 目前，多家制药公司 投入相当多的资源用于合成类似物， 克服细菌耐药性的核糖体抗生素衍生物。 更好地了解核糖体的结构和功能将特别是 重要的抗生素，如大环内酯类，其中耐药性是基于 核糖体结构的变化。