The fidelity of the protein synthetic apparatus is unquestionably of extreme importance in cell function, regulation and replication. Nonetheless errors apparently occur at frequencies several orders of magnitude above those in DNA synthesis. These errors may be involved in progressive diseases such as aging and may also play an important role in natural protein variability. Some viral messages must be """"""""mistranslated"""""""" in order for appropriate proteins to be synthesized.
The aim of the research proposed in this project is to do detailed analysis on the type and frequency of in vivo mistranslational events. Several model proteins, whose genes are carried on bacterial plasmids, will be studied: MS2 coat protein, ornithine transcarbamylase and yeast ribosomal protein L3. These proteins were chosen on the basis of codon usage in their message. Most studies will be done in Escherichia coli but some will also be carried out in the yeast Saccharomyces cerevisiae. The assay to be employed involves amino acid starvation to increase misreading of codons cognate for the deprived amino acid, separation of radiolabeled protein by polyacrylamide gel electrophoresis, electroelution and radiochemical microsequencing. Mistranslation will also be examined in cells experiencing """"""""heat shock."""""""" The internal concentration of tRNA will also be changed by using a tRNA gene which has been placed under the control of a synthetic lac promoter. The influence of codon context will be studied by altering the sequence of the gene(s) by site-directed in vitro mutagenesis. Neighboring bases and codons will be altered as well as potential regions of secondary structure.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025855-09
Application #
3273375
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-12-01
Project End
1991-05-31
Budget Start
1987-06-01
Budget End
1988-05-31
Support Year
9
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Southern Illinois University Carbondale
Department
Type
Schools of Arts and Sciences
DUNS #
939007555
City
Carbondale
State
IL
Country
United States
Zip Code
62901
Fu, C; Parker, J (1994) A ribosomal frameshifting error during translation of the argI mRNA of Escherichia coli. Mol Gen Genet 243:434-41
Ulrich, A K; Li, L Y; Parker, J (1991) Codon usage, transfer RNA availability and mistranslation in amino acid starved bacteria. Biochim Biophys Acta 1089:362-6
Tiedeman, A A; DeMarini, D J; Parker, J et al. (1990) DNA sequence of the purC gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase and organization of the dapA-purC region of Escherichia coli K-12. J Bacteriol 172:6035-41
Precup, J; Ulrich, A K; Roopnarine, O et al. (1989) Context specific misreading of phenylalanine codons. Mol Gen Genet 218:397-401
Goodlove, P E; Cunningham, P R; Parker, J et al. (1989) Cloning and sequence analysis of the fermentative alcohol-dehydrogenase-encoding gene of Escherichia coli. Gene 85:209-14
Parker, J (1989) Errors and alternatives in reading the universal genetic code. Microbiol Rev 53:273-98
Precup, J; Parker, J (1987) Missense misreading of asparagine codons as a function of codon identity and context. J Biol Chem 262:11351-5
Parker, J; Precup, J (1986) Mistranslation during phenylalanine starvation. Mol Gen Genet 204:70-4
Ulrich, A K; Parker, J (1986) Strains overproducing tRNA for histidine. Mol Gen Genet 205:540-5