NUCLEIC ACID PROBES OF RIBOSOME STRUCTURE AND FUNCTION

核糖体是所有细胞中蛋白质生物合成的独特位置，并且 因此，对其结构和功能的详细了解是 对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。 Coli核糖体，这是迄今为止最好的特征，通过研究 很多团体，包括我们自己。然而，考虑到相当大的 在整个进化过程中核糖体结构的保守，我们的结果是 获取也应该有助于理解核糖体与其他 有机体。我们将继续使用放射性、耐光性 具有与单链序列互补的寡核苷酸的衍生物 用于此目的的rRNA序列。这样的探针可以与它们的靶标结合 在完整的核糖体亚基中的序列，并在光解时，结合 转化为相邻的核糖体成分，随后可以被鉴定 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：改变连接耐光性组的间隔物的大小 互补的碱基；不同的耐光性附着部位 以及使用不耐光的寡DNA探针来 监测目标rRNA序列附近的构象变化。 我们还将探索第二种方法的使用，该方法基于 在特定条件下用硫代磷酸盐代替磷酸盐或用4-硫代磷酸盐代替铀 RRNA中的位置。从而将亲电硫化物引入到RNA中 提供用于结合光解性的特定位置(当然， 4-Thiou本身是光敏的)。核糖体的重组 经过修饰的rRNA，然后进行光解，将允许邻近的成分 被确定为寡核苷酸探针无法访问的rRNA位点。 拟议的研究将提供对建设至关重要的信息。 在核糖体的三维结构中，需要 了解核糖体的功能。除了它内在的重要性之外 对生命过程的基本理解，更好地理解 核糖体功能可能会产生重要的治疗后果。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、氯霉素 将核糖体作为其作用部位。对这些核糖体的兴趣 抗生素一直在增长S细菌对β-内酰胺类和 喹诺林类药物已经变得越来越普遍。几家制药公司现在 投入大量资源合成类似物和 核糖体抗生素的衍生物，可克服细菌耐药性。 更好地理解核糖体的结构和功能将特别重要 对抗生素很重要，如大环内酯类，耐药性是基于 核糖体结构的变化。

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.