The experimental idea at the basis of this proposal is that by using both classical techniques of bacterial genetics and the manipulations of modem molecular biology, we can obtain important new ribosomal RNA (rRNA) or rRNA interactive mutants in Escherichia coli as translational suppressors of nonsense or missense mutations, and that those rRNA mutants will provide information about and experimental tools for the further study of the role(s) of rRNA in various functional interactions of the ribosome during translation of the genetic code. In particular, those mutants will allow us to test the hypothesis that specific nucleotides, sequences, structures, regions, or domains of one or more of the rRNAs are involved in particular aspects of specificity and accuracy in polypeptide elongation and termination. Long-range goals include answering the questions: With respect to decoding specificity in the elongation and termination stages of translation, what does rRNA do? Which nucleotides, in which of the three rRNAs, do it? Which nucleotides are involved in specific functional intramolecular interactions within a given rRNA or intermolecular interactions with another rRNA, tRNAs, or other macromolecules? The specific aims are: (1) To target specific rRNA regions, structures, and sites (a) for further characterization of existing mutants and (b) to obtain new kinds of rRNA suppressors and secondary mutations that reverse one or more phenotypes of the primary suppressor mutation, and to characterize the mutant functions and interactions in vivo and in vitro. (2) To reveal and examine in vivo, and ultimately in vitro, specific tRNA-rRNA interactions by obtaining rRNA mutations that specifically affect the action of suppressor tRNA mutants altered in different parts of a tRNA molecule. (3) To devise selections or screens for rRNA mutations that reveal dynamic aspects of translation termination (or elongation) and characterize them in vivo and in vitro. Specific segments of cloned genes will be subjected to random PCR mutagenesis and, when appropriate, specific changes will be introduced by oligonucleotide-directed, site-specific mutagenesis. Cells containing well-characterized trpA missense or nonsense mutations (and, in some cases, mutations in the cat gene), will be transformed with the mutagenized plasmids and spread on plasmid selective media, and the transformants screened for suppression of the reporter gene mutations. Some recipient cells will also contain a suppressor tRNA that is cognate or not to the reporter gene mutation. Information from these studies, which will elucidate the roles of rRNA and the ribosome in translation of the genetic code in single cells and in developmental processes, could lead to development of therapeutic procedures and reagents that interfere with pathogenic bacteria and viruses and that reverse defects in the control of cell growth. In short, our findings touch upon and suggest tools and targets for manipulation of, for example, colds, crops, cattle and cancers.
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