The long range goal of this project still remains to understand the role that polyadenylation plays in post-transcriptional regulation in prokaryotes. Although long considered to be a feature unique to eukaryotes, over the past 10 years it has been shown that polyadenylation is intimately involved in mRNA decay, rRNA processing, tRNA maturation and overall RNA quality contol in Escherichia coli. Of equal importance, it has now been shown that polyadenylation, either by poly(A) polymerase type enzymes or polynucleotide phosphorylase are involved in the post-transcriptional modification of RNA in almost all prokaryotes. In addition, over the past few years it has been shown that eukaryotes contain a nuclear polyadenylation system that closely mimics the E. coli system in that it targets defective Pol II transcripts for degradation. Thus E. coli is an excellent prokaryotic model system for studying this complex system. Based on the progress that we have made during the current grant period, we are now in a position to develop a much more sophisticated understanding of both the biochemical mechanism of polyadenylation and the roles it plays in mRNA decay, tRNA maturation and rRNA processing. Specific experimental approaches include: 1. Analyze the composition and function of the polyadenylation complex in vivo and in vitro;2. Determine the essential requirements for a Rho-independent transcription terminator to function as a polyadenylation signal;3. Determine whether PAP I preferentially polyadenylates full-length or partially degraded transcripts;4. Determine the role of polyadenylation in the maturation of rRNAs and tRNAs;and 5. Analyze the interactions between poly(A) tails and the RNase E-based degradosome in the initiation of mRNA decay. Since polyadenylation appears to be present in a wide range of prokaryotes, including many pathogenic organisms, a more complete understanding of this system provides an opportunity to identify new targets for anti-microbials that will be active against either gram negative or gram positive bacteria.
Project Narrative Although polyadenylation has long been considered to be a unique feature of eukaryotes, over the past ten years it has been shown to be intimately involved in mRNA decay, rRNA processing and tRNA maturation in the model organism Escherichia coli. Of equal importance, it has now been demonstrated that polyadenylation is involved in the post-transcriptional modification of RNAs in almost all prokaryotes. Thus the experiments proposed in this application are designed to develop a much more sophisticated understanding of both the biochemical mechanism of polyadenylation and the roles that it plays in mRNA decay, rRNA processing and tRNA maturation through a combination of biochemical, genetic, and bioinformatic experimental approaches.
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