SITE-SPECIFIC MUTATION IN RRNA AND RIBOSOME FUNCTION

核糖体是所有细胞中合成蛋白质的细胞器。

Ribosomes are the organelles of protein synthesis in all cells. Ribosomes are large ribonucleoprotein complexes composed of many proteins and, usually, three large RNA molecules. The organized interaction of these macromolecules underlies the reactions of protein synthesis (translation) and determines the accuracy of the process. The goal of structural studies on ribosomes is to understand the role of proteins and ribonucleic acid in translation. An understanding of normal protein synthesis may help us to understand aberrant growth processes. We are particularly interested in the role of rRNA in translation. For example, there is evidence that 16S rRNA, a molecule 1542 nucleotides long, performs a number of important roles in ribosome function. It appears that the functional role of different regions of 16S rRNA is determined by the primary structure. We intend to create site-specific base changes in various regions of 16S rRNA to directly test the role of those nucleotides in protein synthesis. To do this, we use a number of deoxyoligonuleotides which differ in sequence by only one base from the wild-type region of 16S rRNA in question. These oligonucleotides will be used as a primer, with the single-strand DNA of the phage M13 as template, which has cloned into it specific regions of 16S rDNA. The in vitro synthesized mutant DNA strand will be used to infect host cells which will generate mutant rDNA molecules. The relevant regions of the mutant rDNA molecules will be ligated into various plasmids which can be grown in E. coli, where the test for the function of the mutated rRNA gene can be carried out. To test for the effect of a mutant rRNA on translational accuracy, the mutant rRNA will be placed in an E. coli carrying a nonsense or frameshift mutation in the LacZ gene and a suppressor t-RNA. Enhancement or restriction of the translation will be a measure of the effect of the mutant rRNA on accuracy of translation. To test for the ability of 30S ribosomes containing mutant rRNA to (1) participate in 30S association with 50S ribosomes, and, (2) to recycle as free 30S ribosomes, plasmids containing mutant rRNA will be used in the "maxicell" system.