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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Modified Research Career Development Award (K04)
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University of Medicine & Dentistry of NJ
Schools of Medicine
United States
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Sipley, J; Stassi, D; Dunn, J et al. (1991) Analysis of bacteriophage T7 gene 10A and frameshifted 10B proteins. Gene Expr 1:127-36
Sipley, J; Dunn, J; Goldman, E (1991) Bacteriophage T7 morphogenesis and gene 10 frameshifting in Escherichia coli showing different degrees of ribosomal fidelity. Mol Gen Genet 230:376-84
Goldman, E; Jakubowski, H (1990) Uncharged tRNA, protein synthesis, and the bacterial stringent response. Mol Microbiol 4:2035-40
Rojiani, M V; Jakubowski, H; Goldman, E (1990) Relationship between protein synthesis and concentrations of charged and uncharged tRNATrp in Escherichia coli. Proc Natl Acad Sci U S A 87:1511-5
Rojiani, M V; Jakubowski, H; Goldman, E (1989) Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli. J Bacteriol 171:6493-502
Brunner, M; di Mayorca, G; Goldman, E (1989) Absence of BK virus sequences in transformed hamster cells transfected by human tumor DNA. Virus Res 12:315-30
Jakubowski, H; Goldman, E (1988) Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae. Mol Cell Biol 8:5166-78
Rojiani, M; Goldman, E (1986) Dependence of MS2 and T4 phage growth upon host amino acid biosynthesis during infections of Escherichia coli. Virology 150:313-7
Ko, J L; Dalie, B L; Goldman, E et al. (1986) Adenovirus-2 early region IA protein synthesized in Escherichia coli extracts indirectly associates with DNA. EMBO J 5:1645-51
Scatina, J; Abdel-Rahman, M S; Goldman, E (1985) The inhibitory effect of Alcide, an antimicrobial drug, on protein synthesis in Escherichia coli. J Appl Toxicol 5:388-94