ORGANIZATION AND FUNCTION OF THE E COLI 16S RIBOSOMAL RN

长期目标是确定三维结构

The long range goal is to determine the three-dimensional structure of the E. coli 16S rRNA in the operating ribosome. This goal includes determining whether changes in the RNA structure occur during protein synthesis and, if this is so, it includes a description of the different states. This approach should identify the loci in the ribosome that define its function. In the present funding period the occurrence of several long-distance contacts within the 16S rRNA in the ribosome will be investigated and the RNA at the active site will be identified. These studies will refine the structure of several areas in an appropriate three-dimensional model of the 16S rRNA and will help determine whether the interactions at these sites are functionally important. This work is important because the E. coli ribosome has been extensively studied and an understanding of protein translation will depend upon the detailed structural analysis of the components and how they physically interact. At the same time, it is becoming clearer that models that describe translation and the regulation of translation in eubacteria will also be applicable to eukaryotes. These approaches will be taken: (1) Determine whether there are differences in the long distance interactions in the 16S rRNA which are correlated to the functional state of the subunit. Psoralen photochemical crosslinking and analysis by denaturing gel electrophoresis will be used to detect intra RNA contacts. It may be necessary to direct psoralen monoadducts to specific sites in order to gain sensitivity and to avoid killing the biological activity of the RNA. (2) Identify the position and boundaries of the active sites in the 30S ribosome, by using synthetic or natural mRNA carrying photochemical reagents. (3) Modify the ribosomal RNA and test for structural/functional correlations: characterize the structure of altered 16S rRNA made by Dr. A. Dahlberg and co-workers (Brown University); alter the 16S rRNA by attaching DNA oligonucleotides at several chosen sites to selectively affect its functional activity; make 16S rRNA by in vitro transcription and reconstitute it into an active subunit. If the last experiment is successful, then selectivity alter regions within the gene by in vitro manipulations.