NUCLEIC ACID PROBES OF RIBOSOMAL STRUCTURE AND FUNCTION

核糖体是所有细胞中蛋白质生物合成的独特位点，因此对其结构和功能的详细了解对于在分子水平上更普遍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的重要性外，更好地了解核糖体的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、氯霉素等都以核糖体为作用位点。随着细菌对-内酰胺类和喹啉类药物的耐药性越来越普遍，对这些核糖体抗生素的兴趣也越来越大。几家制药公司目前正投入大量资源合成核糖体抗生素的类似物和衍生物，以克服细菌耐药性。更好地了解核糖体结构和功能对抗生素尤其重要，如大环内酯类抗生素，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，这是迄今为止许多小组研究中最具特色的，包括我们自己的研究。然而，考虑到核糖体结构在整个进化过程中的相当大的保守性，我们获得的结果也应该有助于理解其他生物的核糖体。本提案的总体目标是描述核糖体在其功能周期的特定步骤中所经历的构象变化，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位点形成明确的光交联来实现这一点，这些位点对核糖体的结构和功能具有重要意义，利用光交联过程的固有能力来取样溶液中的所有构象。这种交联将在不同的功能状态下形成，在野生型和突变型核糖体中，在存在和不存在抗生素的情况下。这种交联所代表的结构约束，以及其他方法产生的约束，将用于模拟特定功能状态下的核糖体结构，使用70S核糖体的晶体结构和30S和SOS亚基作为初始结构。作为我们的主要方法，我们将继续和完善使用具有与rRNA序列互补序列的放射性、光稳定的寡核苷酸衍生物（phont）。这些探针在完整的核糖体亚基中结合到它们的目标序列上，并且在光解作用下，结合到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，基于在完整rRNA中引入特定位点的光致性（IPHOR -完整的光致性RNA），以获得phont无法进入或使用phont诱导主要构象变化的rRNA位点的类似信息。

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.