Translational Control of mRNA processing in Escherichia coli

University Of Washington, Seattle WA

Investigators

Abstract

Moseley The project will investigate a novel mechanism of mRNA processing in Escherichia coli. A polycistronic message encoding assembly and structural elements of E. coli adhesive fimbriae is cleaved at a specific site upstream of the gene encoding the major structural element. The cleavage generates a very stable message encoding the major fimbrial subunit. mRNA cleavage is coupled to translation of an open reading frame, daap, which spans the cleavage site. The project will test the hypothesis that the nascent DaaP peptide interacts with components associated with the ribosome, and that the interaction leads to cleavage of the daa mRNA through the action of a component associated with the ribosorne. The two major objectives of the project are: 1) a genetic analysis of daa MRNA processing, and 2) a biochemical analysis of the processing event. The genetic analysis involves two approaches. In the first approach, E. coli mutants which fail to process daa MRNA will be isolated and characterized, providing information about specific E. coli gene products involved in the cleavage reaction. The second approach will seek to exploit the observation that processing does not occur in Pseudomonav aeruginosa. E. coli chromosomal libraries will be screened in P. aeruginosa for acquisition of daa MRNA processing activity. The biochemical characterization of the processing reaction focuses on the ribosorne. A cell free processing reaction will be developed and optimized utilizing E. coli S30 extracts and purified components. Fractions and purified components from P. aeruginosa and E. coli will be combined in a cell free processing assay to identify E. coli components involved in mRNA cleavage. Ribosomal and soluble components from wild type E. coli will be combined with extracts from non-processing mutant strains to localize and identify complementing activities. The research will also determine if the nascent DaaP peptide induces stalling of the ribosome in association with the cleavage event. The action of a nascent peptide in mRNA processing suggests an important new role for the ribosome in the regulation of gene expression. The objectives outlined above seek to explore the molecular interactions involved in this activity through genetic and biochemical experimental approaches. These investigations should provide a new understanding of a novel mechanism of gene regulation at the post-transcriptional level.