This application proposes the use of missense suppression to study the roles of transfer RNAs (tRNAs), messenger RNA (mRNA) codon context, and ribosomal RNAs (rRNAs) in codon recognition during polypeptide chain elongation. The well-characterized, suppressible missense codons that are to be employed are in the trpA gene of Escherichia coli. The experiments that are described aim at answering the following six questions: (a) Do isoacceptor missense suppressor tRNAs read a given codon differently at different positions in trpA? (b) Can a given missense suppressor tRNA be further altered mutationally to result in an altered efficiency of codon recognition, for both cognate and non-cognate codons? (c) Can in vivo evidence be obtained that there is interaction between two tRNAs on the ribosome? (d) How far upstream or downstream from a given codon can mutations occur that affect the translation of that codon? (e) What effect, if any, on translation of a given codon, will result from specifically and systematically changing each of the three nucleotides immediately preceding or following the codon? And finally, (f) Can mutationally altered ribosomal RNAs function as missense suppressors? The effects of mutations in tRNA, mRNA, or rRNA on missense suppression will be quantitated by assaying the trpA gene product. The necessary mutations either already exist or will be obtained by standard genetic and biochemical technology either in vivo or in vitro. The resulting understanding of molecular mechanisms involved in codon recognition in this """"""""simple"""""""" system will be most valuable in interpreting the significance of variations of tRNAs, and other translational components, observed in differentiating cells and malignant cells, in different organs, and in response to viruses, carcinogens, and chemotherapeutic agents. In addition, such fundamental knowledge is important for attempts to accurately engineer and produce medically and industrially important proteins.
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