ANALYSIS OF TRNA CODING IN PROTEIN IN SYNTHESIS

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 date4/1/801/31/93

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $124,064.00
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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