A molecular genetic approach that employs translational suppression is being taken to reveal new ribosomal RNA (rRNA) determinants of specificity in translation and characterize structural determinants of specific functional interactions between rRNA and other translational macromolecules. Long-range goals include finding answers to the following questions: With respect to decoding specificity in the elongation and termination steps of translation in E. coli, what does rRNA do? Which nucleotides, in which of the three rRNAs, do it? Which nucleotides are involved in specific functional interactions with transfer RNAs (tRNAs) or with external insults such as virus-encoded antisense RNAs? The specific aims are: (1) To select for new kinds of rRNA translational suppressors, determine their mutational changes, and characterize their altered translational functions in vivo. (2) To select for rRNA mutants that interact specifically, in decoding, with one of two particular parts of a tRNA molecule, the acceptor stem or anticodon loop, determine their DNA sequence changes, and study the precise interactions with that part of the tRNA. (3) To characterize further the suppressor and inhibitory actions of the RNA encoded by a cloned phage lambda bar segment, as to its apparent involvement in peptide chain termination and its possible interaction or co-functioning with the RNA of the small subunit of the ribosome. For the most part, cloned rRNA genes will be subjected to random mutagenesis in vivo. E. coli cells containing well-characterized trpA mutations will be transformed with the mutagenized plasmid and spread on media to select simultaneously for plasmic acquisition and suppression of the trpA mutations. Some recipient cells will also contain a suppressor tRNA that in the presence of wild-type rRNA does not suppress the particular trpA mutation. Information from these studies could lead to development of therapeutic procedures that discriminate against detrimental viruses or bacteria that depend upon specialized translational events for their propagation. It will also be most important for attempts to accurately engineer and produce medically and industrially important proteins.
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