ANALYSIS OF TRNA CODING BY PROTEIN SYNTHESIS IN VITRO

Project Details

Description

We proposed to study the following areas: (1) Examination of rate-limiting
elongation in Escherichia coli as means of controlling protein synthesis,
including looking at the mechanism, asking if a postulated mathematical
description is general for other messages and codons, varying the total
tRNA concentration in cells to see what effects this has and if uncharged
tRNA plays a role in regulation, testing mutant tRNAs for translational
efficiency, determining what amino acid is incorporated when rate-limiting
an elongation step, seeing if methods of perturbing fidelity affect
rate-limiting elongation, analyzing the partial growth defect of phage MS2
on Su+6 cells, characterizing a cloned gene from Su+6 cells which led to a
bizarre phenotype, studying the mechanism by which tRNA deprivation
inhibits phage MS2 RNA synthesis, and examining the mechanism responsible
for variable tRNA levels in tRNA synthetase mutant. (2) Determination of
codon recognition by E. coli tRNA isoacceptors using protein synthesis in
vitro directed by sequenced mRNA or DNA (in a coupled system).
Site-specific incorporation of a radioactive amino acid into a known
position in a protein encoded by a known codon will permit a quantitative
assessment of specific tRNA function, including tRNA-Ser and tRNA-Leu
species, possible effects of mRNA superstructure, reading context, and
preferences for one isoacceptor over another. (3) Effect of ribosomal
fidelity mutants, strains which fail to grow tRNA mutants of phage T4,
relaxed mutants, and tRNA from cells with mutations in tRNA modification
enzymes. These experiments will also test our model of a "translational
hierarchy" of tRNAs. (4) Exploration of misreading in vitro under
tRNA-dependent conditions, including determining substituted amino acids,
testing amino acid starvation in vitro, and mixing extracts of ribosomal
and tRNA-synthetase deficient mutants. The experiments proposed in this
research program should further our understanding of tRNA structure and
function, and of the mechanism and regulation of protein synthesis, with
possible implications for our understanding of aging and/or cancer.
StatusFinished
Effective start/end date4/1/871/1/90

Funding

  • National Cancer Institute
  • National Cancer Institute
  • National Cancer Institute

ASJC

  • Genetics

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