The object of this project is: to understand how the ribosome participates in the reactions of protein synthesis. The ribosome is a large complex ribonucleoprotein particle. The rRNA plays an important role in its function. This includes base-pairing with mRNAs during translational initiation in prokaryotic systems, and possibly, other 'catalytic' roles in the reactions of protein synthesis. These proposed interactions may involve a small region of primary structure or regions of RNA far apart on the primary structure, brought physically close by the higher order structure of the ribosome. To investigate the relationship between the primary structure of rRNA and various functional roles, binding or catalytic, we are utilizing the procedures of site-specific mutagenesis. Base changes are made to test if (a) the mutant rRNA will be incorporated into ribosomes and, if incorporated, (b) will they affect a specific reaction of protein synthesis. Ideally a single base change in an important functional region will interfere with a unique stage of protein synthesis. Ultimately, the identification of all bases involved in a unique function will establish the "active" site for that reaction. Specific aims: 1. Production of site-specific mutations in 16S and 23S rRNA at putative functional sites using a plasmid coded rrnB operron. 2. Synthesis of mutant ribosomes. Mutant rDNA molecules in different plasmids will be transformed into E. coli, where specific in vivo functional tests will be carried out. Mutant rRNA molecules will also be studied when under control of a number of non-rRNA promoters which allows the controlled expression of mutant rRNA molecules and allows for the study of mutations which when uncontrolled, are lethal to the cell. 3. Structural and functional assay of mutant ribosomes. A. In vivo tests will include: (i) Tests for the effects on translation. For example the mutant rRNA is introducted into an E. coli strain carrying a nonsense mutation in the Lac Z gene and a suppressor t-RNA. Alteration in the level of enzyme will be a measure of the effect of the mutant rRNA on translation. (ii) Association- Dissociation reaction: using the "maxicell" system we will determine if mutant 30S ribosomes will form 70S ribosomes, form polysomes, and recycle as cell-free extracts of E. coli and testing for their ability to carry out defined reactions of parotein synthesis.
