Project Details

Description

The experiments proposed are designed to critically test and expand various aspects of our working model of what happens to the elongation process in bacteria when ribosomes encounter a rate-limiting elongation step. Section One is concerned with the events on the ribosome when it encounters a hungry codon. Does uncharged tRNA play a role? How general are the effects we have been studying? Can we show premature termination and peptidyl-tRNA release? Do the mathematics suggested by our initial studies hold up for other codons or messages? Do tRNA mutations or methods of perturbing fidelity affect our results. Are translational errors specific or heterogenous? Can we resolve anomalies encountered in studying phage MS2 under these conditions, such as unexpected effects on phage RNA synthesis, and partial inhibition by a tRNA mutation (Su+6) on MS2 growth? Section Two is concerned with the fidelity of translation at the hungry codon. Do frameshifts occur, and what governs the process? Can amino acid substitutions be exacerbated and studied in vitro? Can we confirm earlier results of ours in which we isolated a pleiotropic suppressor (presumably an "error function") from Su+6 cells? Sections Three and Four address codon recognition itself, with the intent of establishing a translational hierarchy of tRNAs able to respond to a given codon, as well as to examine aspects of the role of the ribosome in codon recognition. Section Three sets up the system by examining individual tRNA-Ser and tRNA-Leu isoacceptor species for codon recognition by translation of a natural mRNA. Site-specific incorporation of a radioactive amino acid into a known position in a protein, encoded by a known codon, permits a quantitative assessment of tRNA function. We will also assess possible effects of mRNA superstructure, reading context effects, and possible preferences for one tRNA isoacceptor over another tRNA in competition experiments. Section Four exploits the system further, by examining mutant ribosomes and mutants tRNAs, and seeing how these mutations affect codon recognition. Mutations to be studied include ribosomal "flexibility" mutants, relaxed strains, and tRNA modification mutants. Phage T4 tRNA deletion mutants, and strains which fail to grow them, will also be examined. It is hoped that the experiments proposed in this application will lead to a significant contribution in our understanding of various aspects of tRNA structure and function, and of the mechanism and regulation of protein synthesis. The results of this research, in addition to expansion of our basic knowledge, may also have practical implications in our understanding of cancer, of aging processes, as well as implications for genetic engineering technology.
StatusFinished
Effective start/end date1/1/901/31/93

Funding

  • National Institute of General Medical Sciences

ASJC

  • Genetics

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