The long range goal of this project is to understand how the process of polyadenylation helps regulate RNA decay in prokaryotes. Long considered a feature unique to eukaryotic organisms, recent work has demonstrated unequivocally that polyadenylation occurs in bacteria and is an integral part of the post-transcriptional machinery that degrades both mRNA and rRNA. We hypothesize that polyadenylation helps cells rapidly adapt to changes in their environment by targeting certain mRNA and rRNA species for decay. In this fashion, for example, a cell can undergo a smooth transition from logarithmic growth to stationary phase. In order to test this hypothesis, we plan to: 1. Determine how polyadenylation activity varies when cells have to adapt to different growth conditions; 2. Determine the physiological consequences of altering poly(a) levels within the cell; 3. Determine the mechanism of polyadenylation in the absence of poly(A)polymerase I; 4. Purify and characterize potential multi-protein complexes that bind to 3' termini of mRNAs using tagged poly(A) polymerase I, Rnase II and polynucleotide phosphorylase; 5. Determine the biochemical components of poly() dependent mRNA decay using our in vitro assay system; and 6. Determine if there are polyadenylation target sequences. With the experiments described in this application, we hope to obtain a more sophisticated understanding at the molecular level of the function of polyadenylation in prokaryotes. If normal polyadenylation is required for bacterial cells to adapt to changing growth environments, components of the system may prove to be attractive targets for new antimicrobials.

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National Institute of General Medical Sciences (NIGMS)
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Molecular Biology Study Section (MBY)
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University of Georgia
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Mildenhall, Kristen B; Wiese, Nicholas; Chung, Daewhan et al. (2016) RNase E-based degradosome modulates polyadenylation of mRNAs after Rho-independent transcription terminators in Escherichia coli. Mol Microbiol 101:645-55
Mohanty, Bijoy K; Petree, Jessica R; Kushner, Sidney R (2016) Endonucleolytic cleavages by RNase E generate the mature 3' termini of the three proline tRNAs in Escherichia coli. Nucleic Acids Res 44:6350-62
Dubnau, David (2015) Regulation by the modulation of gene expression variability. J Bacteriol 197:1974-5
Kushner, Sidney R (2015) Polyadenylation in E. coli: a 20 year odyssey. RNA 21:673-4
Mohanty, Bijoy K; Kushner, Sidney R (2014) In vivo analysis of polyadenylation in prokaryotes. Methods Mol Biol 1125:229-49
Agrawal, Ankit; Mohanty, Bijoy K; Kushner, Sidney R (2014) Processing of the seven valine tRNAs in Escherichia coli involves novel features of RNase P. Nucleic Acids Res 42:11166-79
Mohanty, Bijoy K; Kushner, Sidney R (2013) Deregulation of poly(A) polymerase I in Escherichia coli inhibits protein synthesis and leads to cell death. Nucleic Acids Res 41:1757-66
Mohanty, Bijoy K; Maples, Valerie F; Kushner, Sidney R (2012) Polyadenylation helps regulate functional tRNA levels in Escherichia coli. Nucleic Acids Res 40:4589-603
Mohanty, Bijoy K; Kushner, Sidney R (2011) Bacterial/archaeal/organellar polyadenylation. Wiley Interdiscip Rev RNA 2:256-76
Stead, Mark B; Marshburn, Sarah; Mohanty, Bijoy K et al. (2011) Analysis of Escherichia coli RNase E and RNase III activity in vivo using tiling microarrays. Nucleic Acids Res 39:3188-203

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