Our long-term objective is to understand the role played by ribosomal RNA in protein synthesis. The fundamental processes involved in protein synthesis are believed to be the same in all organisms and several studies have indicated direct involvement of conserved ribosomal RNA sequences in translation. Small-subunit rRNAs all contain three single-stranded sequences which are among the most highly conserved sequences in nature. The goal of this project is to determine the functional significance of these highly conserved, single-stranded sequence within small-subunit ribosomal RNAs. Phylogenetic analyses and our previous results suggest that base pairing at specific residues between two of these highly conserved sequences is required for ribosome function. However, physical and biochemical studies, indicate that these residues are unpaired in nontranslating ribosomes. We will test the hypothesis that ribosome function in vivo is dependent upon the ability of these residues to transietly base pair. The presence of multiple rRNA genes in most organisms has complicated genetic analysis of ribosome function. To circumvent these problems we have constructed a genetic system which does not interfere with normal cellular function. In this system the chloramphenicol acetyltransferase (CAT) reporter message is translated exclusively by plasmid encoded ribosomes which cannot translate normal cellular messages. Consequently, cells containing this construct are chloramphenicol resistant and the level of this resistance is dependent upon the amount of functional CAT protein produced by plasmid derived ribosomes. Thus, deleterious rRNA mutations in plasmid encoded ribosomes will inhibit translation of only the CAT message and therefore decrease chloramphenicol resistance without affecting translation of other cellular messages. We will construct ribosomal RNA mutations which affect base pairing among these sequences, measure their effect on translation and characterize the step(s) during translation which is affected.
Our specific aims are; 1. Construct mutations at the proposed sites of interactions. Mutations will be constructed which disrupt putative base pairing. Function in mutants with the potential to base pair will be compared to those which cannot pair. 2. Create additional potential sites of interaction. Nucleotides neighboring those implicated in pairing will be mutated to create additional sites of interaction and the effects of these mutations will be determined. 3. Select and identify second site revertants. Nonfunctional or partially functional ribosomal RNA mutants will be used to select functional, second- site revertants. 4. Biochemical analysis of mutant ribosome function. Selected mutants from each class will be analyzed to determine which aspect of protein synthesis is affected by the mutation. These analyses will reveal if base pairing between conserved sequences plays a role in translation, the specific step at which pairing is essential, and contribute to our understanding of rRNA function.
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