We propose to study the following areas: (1) Examination of rate-limiting elongation in Escherichia coli as a 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 Su6+ cells, characterizing a cloned gene from Su6+ cells which led to a bizarre phenotype, completing analysis of our new method for measuring aminoacylated tRNA levels in vivo, and examining a new feedback loop mechanism from the rate of protein synthesis to tRNA aminoacylation levels. (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 mutations on tRNA coding properties, including ribosomal """"""""flexibility"""""""" mutants, strains which fail to grow tRNA mutants of phage T4, and relaxed mutants; these experiments will also test our model of """"""""translational hierarchy"""""""" of tRNAs. (4) Codon recognition properties of mutant tRNAs and nutritionally obtained undermodified tRNAs, which will help assess the contribution of various modifications to function. (5) 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 application 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027711-06
Application #
3274936
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-04-01
Project End
1987-01-31
Budget Start
1985-09-01
Budget End
1987-01-31
Support Year
6
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Type
Schools of Medicine
DUNS #
605799469
City
Newark
State
NJ
Country
United States
Zip Code
Goldman, E; Rosenberg, A H; Zubay, G et al. (1995) Consecutive low-usage leucine codons block translation only when near the 5' end of a message in Escherichia coli. J Mol Biol 245:467-73
Gao, W; Goldman, E; Jakubowski, H (1994) Role of carboxy-terminal region in proofreading function of methionyl-tRNA synthetase in Escherichia coli. Biochemistry 33:11528-35
Sipley, J; Goldman, E (1993) Increased ribosomal accuracy increases a programmed translational frameshift in Escherichia coli. Proc Natl Acad Sci U S A 90:2315-9
Rosenberg, A H; Goldman, E; Dunn, J J et al. (1993) Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system. J Bacteriol 175:716-22
Jakubowski, H; Goldman, E (1993) Synthesis of homocysteine thiolactone by methionyl-tRNA synthetase in cultured mammalian cells. FEBS Lett 317:237-40
Jakubowski, H (1993) Energy cost of proofreading in vivo: the charging of methionine tRNAs in Escherichia coli. FASEB J 7:168-72
Kim, H Y; Ghosh, G; Schulman, L H et al. (1993) The relationship between synthetic and editing functions of the active site of an aminoacyl-tRNA synthetase. Proc Natl Acad Sci U S A 90:11553-7
Jakubowski, H (1993) Proofreading and the evolution of a methyl donor function. Cyclization of methionine to S-methyl homocysteine thiolactone by Escherichia coli methionyl-tRNA synthetase. J Biol Chem 268:6549-53
Jakubowski, H; Goldman, E (1993) Methionine-mediated lethality in yeast cells at elevated temperature. J Bacteriol 175:5469-76
Cai, X Y; Jakubowski, H; Redfield, B et al. (1992) Role of the metF and metJ genes on the vitamin B12 regulation of methionine gene expression: involvement of N5-methyltetrahydrofolic acid. Biochem Biophys Res Commun 182:651-8

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