STRUCTURE AND MECHANISM OF RIBOSOMAL RNA IN THE RIBSOME

核糖体是一个重要的研究对象，因为它执行一个普遍的和关键的生化过程，其分子细节尚不清楚。我们对RNA催化能力的理解以及我们对RNA和核糖核蛋白结构的一般知识将通过对rRNA机制的阐明而大大增加。所提出的研究的一个重要方面是，许多抗生素在核糖体上发挥其活性。如果要合理设计合成抗生素，有必要了解这些相互作用的性质及其对核糖体的影响。我们已经制定了策略来确定16S rRNA中反映其构象状态的分子内光和光化学交联的位置和频率。我们知道16SrRNA会受到特定的和离散的构象变化的影响，这取决于它的相互作用和环境。具体目的是获得有关rRNA结构细节的更多信息，确定rRNA构象在控制tRNA结合和易位中的作用，并确定抗生素如何干扰蛋白质合成。这些实验所需的技术是可用的，具体的目标将彼此独立地进行。工作的具体目的是：(1)完成交联实验，完善16S rRNA在30S亚基和70S核糖体中的rRNA处理区模型；(2)确定四环素和大霉素结合过程中发生的结构变化的细节，并确定这些变化如何干扰tRNA结合和易位过程；(3)确定16S rRNA构象如何决定tRNA结合和tRNA易位；(4)确定16S rRNA在885-914区间内二级结构变化的全局结构后果，确定氨酰基- trna . eftu的关联。GTP三元配合物影响16S rRNA结构，它们决定了不同构象如何影响氨基酰基- trna结合。

The ribosome is an important subject for investigation because it performs a universal and key biochemical process for which the molecular details are not known. Our understanding of the catalytic capabilities of RNA and our general knowledge of RNA and ribonucleoprotein structure will be greatly increased by an elucidation of the rRNA machinery. An important aspect of the proposed studies is that many antibiotics exert their activities on the ribosome. It is necessary to understand the nature of these interactions and their effects on the ribosome if there is to be rational design of synthetic antibiotics. We have developed strategies to determine the locations and frequencies of intramolecular photo- and photochemical crosslinks in the 16S rRNA which reflect its conformation status. We know that the 16SrRNA is subject to specific and discrete conformation changes that depend on its interactions and environment. The specific aims are designed to obtain more information about the details of the rRNA structure, to determine the role of the rRNA conformation in controlling tRNA binding and translocation, and to determine how antibiotics interfere with protein synthesis. The techniques needed for these experiments are available and the specific aims will proceed independent of one another. The specific aims of the work are: (1) Complete crosslinking experiments to refine a model for the rRNA handling region of the 16S rRNA in the 30S subunit and 70S ribosome; (2) Determine the details of the structural changes that occur during tetracycline and spectinomycin binding and determine how these changes interfere with tRNA binding and the translocation process; (3) Determine how the 16S rRNA conformation determines tRNA binding and tRNA translocation; and (4) Determine the global structural consequences of the 16S rRNA secondary structure change in the interval 885-914 that occurs in the 16S rRNA, determine how association of aminoacyl-tRNA.EFTu.GTP ternary complex affects 16S rRNA structure, and them determine how the different conformations affects aminoacyl-tRNA-binding.