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Title:Regulation of the Mannose PTS Operon by the small RNA SgrS
Author(s):Rice, Jennifer
Director of Research:Vanderpool, Carin K.
Doctoral Committee Member(s):Farrand, Stephen K.; Cronan, John E.; Miller, Charles G.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):small RNA (sRNA)
sugar transport-related sRNA (SgrS)
glucose-phosphate stress
Ribonucleic acid (RNA)
Abstract:A number of bacterial small RNAs (sRNAs) act as global regulators of stress responses by controlling expression of multiple genes. The sRNA SgrS is expressed in response to glucose-phosphate stress, a condition associated with disruption of glycolytic flux and accumulation of sugar-phosphates. SgrS has been shown to stimulate degradation of the ptsG mRNA, encoding the major glucose transporter. This study demonstrates that SgrS regulates the genes encoding the mannose and secondary glucose transporter, manXYZ. Analysis of manXYZ mRNA stability and translation in the presence and absence of SgrS indicate that manXYZ is regulated by SgrS under stress conditions and when SgrS is ectopically expressed. In vitro footprinting and in vivo mutational analyses showed that SgrS base pairs with manXYZ within the manX coding sequence to prevent manX translation. Regulation of manX did not require the RNase E degradosome complex, suggesting that the primary mechanism of regulation is translational. An Escherichia coli ptsG mutant strain that is manXYZ(+) experiences stress when exposed to the glucose analogs α- methyl glucoside or 2-deoxyglucose. A ptsG manXYZ double mutant is resistant to the stress, indicating that PTS transporters encoded by both SgrS targets are involved in taking up substrates that cause stress. We further demonstrate that SgrS binds to two sites on the manXYZ mRNA to coordinately down-regulate translation of all the cistrons, a mechanism reminiscent of that used by the eukaryotic miRNA, lin-4 for regulation of the lin-14 mRNA. We tested the hypothesis that regulation of manY and manZ is dependent on SgrS:manX base pairing and subsequent manXYZ mRNA degradation. Contrary to the hypothesis, we show that SgrS repression of iii manY and manZ translation can be decoupled from SgrS-dependent manXYZ mRNA degradation. Furthermore, SgrS regulates manY translation by a mechanism that is independent of SgrS:manX mRNA interactions. Instead, translational regulation of manY depends on SgrS pairing with sequences in the manX-manY intergenic region, upstream of the manY ribosome binding site. Mutational analysis suggests that while the SgrS-manY interaction is sufficient to stimulate some degradation of the manXYZ mRNA, pairing with both sites is required for maximal degradation of the manXYZ mRNA. Additional SgrS candidate-mRNA targets are also investigated.
Issue Date:2012-02-01
Genre:Dissertation / Thesis
Rights Information:Copyright 2011 Jennifer Rice
Date Available in IDEALS:2012-02-01
Date Deposited:2011-12

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