Protein biosynthesis involves the participation of a large number of species. Of particular interest are those protein factors which are involved in the itiation of translation. Initiation factor 3 (IF3) is crucial for this process by ensuring that a pool of free 30S ribosomal subunits is available and by stimulating the func- tional interaction of mRNA with the 30S subunit. Several questions remain outstanding with regard to the biosynthesis of IF3 and its functional interaction with the ribosome. The gene encoding IF3, infC, has been cloned in this laboratory and several promoters which control the expression of the gene have been identified. Promoter fusion and S1 nuclease mapping studies will be carried out in order to analyze the activity and regulation of transcription which is initiated from each of these promoters. These will include an investigation of the growth rate regulation of infC expression and the possible involvement of stringent control. Little is currently known regarding the structure-function relationships of IF3. Mutant forms of IF3 will be produced by both deletion and site-specific mutagenesis. The biologicaL activities of the mutant proteins will be determined in a variety of functional assays. This should provide information on the composition of the active site of IF3. Previous studies in this laboratory have shown that IF3 and its gene have been evolutionarily conserved. The infC-like genes and IF3 from several organisms other than E. coli will be isolated sequenced and functionally characterized. This data should facilitate identification of those domains of IF3 which have been conserved and are presumably required for function. The protein components of the ribosome binding site for IF3 have been identified but this has not been accomplished for the 16S RNA portion of the site. This will be analyze by the construction of 30S ribosomal subunits with site-specific mutations in the 16S RNA. The ability of IF3 to bind to these mutant subunits will be assessed. The proposed experiments will clarify aspects of the regulation of IF3 biosynthesis provide a more comprehensive description of the structure-function relationships of and reveal the involvement of specific 16S RNA sequences in IF3 binding. Since IF3 plays central role in the initiation of protein synthesis, these studies will, in turn allow for a more complete understanding of the molecular mechanisms of this critical step in gene expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM029265-08
Application #
3276829
Study Section
Biochemistry Study Section (BIO)
Project Start
1980-09-01
Project End
1993-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
8
Fiscal Year
1988
Total Cost
Indirect Cost
Name
New York Medical College
Department
Type
Schools of Medicine
DUNS #
City
Valhalla
State
NY
Country
United States
Zip Code
10595
Maar, Dianna; Liveris, Dionysios; Sussman, Jacqueline K et al. (2008) A single mutation in the IF3 N-terminal domain perturbs the fidelity of translation initiation at three levels. J Mol Biol 383:937-44
Ganoza, M C; Aoki, H; Kozieradzki, I et al. (1993) Reconstruction of translation. Evidence for the involvement of the rescue protein in the association/dissociation of ribosomal subunits. Eur J Biochem 217:839-47
Liveris, D; Schwartz, J J; Geertman, R et al. (1993) Molecular cloning and sequencing of infC, the gene encoding translation initiation factor IF3, from four enterobacterial species. FEMS Microbiol Lett 112:211-6
De Bellis, D; Liveris, D; Goss, D et al. (1992) Structure-function analysis of Escherichia coli translation initiation factor IF3: tyrosine 107 and lysine 110 are required for ribosome binding. Biochemistry 31:11984-90
Liveris, D; Klotsky, R A; Schwartz, I (1991) Growth rate regulation of translation initiation factor IF3 biosynthesis in Escherichia coli. J Bacteriol 173:3888-93
De Bellis, D; Schwartz, I (1990) Regulated expression of foreign genes fused to lac: control by glucose levels in growth medium. Nucleic Acids Res 18:1311
Santer, M; Bennett-Guerrero, E; Byahatti, S et al. (1990) Base changes at position 792 of Escherichia coli 16S rRNA affect assembly of 70S ribosomes. Proc Natl Acad Sci U S A 87:3700-4
Goodman, R; Schwartz, I (1988) Kinetic analysis of an E.coli phenylalanine-tRNA synthetase mutant. Nucleic Acids Res 16:7477-86
Wertheimer, S J; Klotsky, R A; Schwartz, I (1988) Transcriptional patterns for the thrS-infC-rplT operon of Escherichia coli. Gene 63:309-20
Klotsky, R A; Schwartz, I (1987) Measurement of cat expression from growth-rate-regulated promoters employing beta-lactamase activity as an indicator of plasmid copy number. Gene 55:141-6

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