The long-range goal of this project is to understand the role that polyadenylation plays in prokaryotic post-transcriptional regulation. Long considered a feature unique to eukaryotic organisms, recent work has demonstrated unequivocally that polyadenylation plays an integral role in both mRNA decay, rRNA processing and rRNA degradation. We have hypothesized that polyadenylation helps cells rapidly adapt to changes in their environment by targeting certain mRNA and rRNA species for decay. Since poly(A) polymerase homologues are found in a large number of prokaryotes, Escherichia coli is an excellent model system for studying this complex system. The experiments described here are designed to help elucidate the molecular mechanism of polyadenylation and to derive a more sophisticated understanding of its function in the growth and survival of the cell. Specific lines of approach include: 1. Analyze the interactions of poly(A) polymerase I, polynucleotide phosphorylase and the Hfq protein with its mRNA substrates; 2. Determine the essential features of mRNA polyadenylation signals; 3. Clone and characterize the stationary phase-specific poly(A) polymerase; 4. Determine the function of polynucleotide phosphorylase in the polyadenylation and mRNA decay pathways; 5. Determine if polyphosphate kinase (Ppk) plays a role in regulating the polyadenylation pathway; 6. Determine whether polyadenylation affects how cells adapt to different growth conditions; 7. Identify and characterize the role of potential poly(A) binding proteins, including CspE; and 8. Determine if polyadenylation affects translation efficiency. Since polyadenylation seems to function very differently in E. coli compared to eukaryotic organisms, it is important to develop a better understanding of how this system works. An additional potential benefit from this work is the possible identification of new targets for antimicrobials.
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