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，其分子细节是未知的。通过对rRNA机制的阐明，我们对RNA催化能力的理解以及对RNA和核糖核蛋白结构的一般知识将大大增加。拟议研究的一个重要方面是许多抗生素对核糖体发挥其活性。如果要合理设计合成抗生素，就必须了解这些相互作用的性质及其对核糖体的影响。我们已经制定了战略，以确定的位置和频率的分子内的光和光化学交联的16 S rRNA，反映其构象状态。我们知道，16 SrRNA受到特定的和离散的构象变化，这取决于它的相互作用和环境。具体目标是获得更多关于rRNA结构细节的信息，确定rRNA构象在控制tRNA结合和易位中的作用，以及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，具体目标将相互独立地进行。本工作的具体目标是：（1）完成交联实验，以完善30 S亚基和70 S核糖体中16 S rRNA处理区的模型;（2）确定四环素和壮观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16 S rRNA构象如何决定tRNA结合和tRNA移位;和（4）确定16 S rRNA二级结构在885-914区间发生变化的总体结构后果，确定氨酰-tRNA·EFTu·GTP三元复合物的缔合如何影响16 S rRNA结构，它们决定了不同的构象如何影响氨酰-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.