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Title:Target regulation and prioritization by the small RNA SgrS in Escherichia coli
Author(s):Bobrovskyy, Maksym
Doctoral Committee Chair(s):Vanderpool, Carin K.
Doctoral Committee Member(s):Shisler, Joanna L.; Cronan, John E.; Gardner, Jeffrey F.
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Small RNA
SgrS
Glucose-phosphate stress response
Post-transcriptional regulation
Bacterial gene regulation
Bacterial physiology
Abstract:Regulation of gene expression by small non-coding RNAs is ubiquitous in all domains of life. In bacteria, small RNAs are known regulators of various stress responses. Diverse mechanisms employed by small RNAs demonstrate multi-faceted nature of gene regulation, tailored to respond to stress with optimal efficiency. Likewise, the Escherichia coli small RNA SgrS controls a response to metabolic stress that occurs upon cytoplasmic accumulation of glucose-phosphates due to mutations in glycolysis (e.g. in pgi) or when cells take up glucose-analogs α-methyl-D-glucoside (αMG) and 2-deoxyglucoside (2DG). SgrS base pairs with and represses translation of ptsG and manXYZ mRNAs, which encode sugar transporters, and activates translation of yigL mRNA, encoding a sugar phosphatase. In this study, transcriptomic analyses along with genetics and biochemistry defined four new direct targets of E. coli SgrS. These new target mRNAs, asd, adiY, folE and purR, encode transcription factors or enzymes of diverse metabolic pathways, including aspartate semialdehyde dehydrogenase, arginine decarboxylase gene activator, GTP cyclohydrolase I and a repressor of purine biosynthesis, respectively. SgrS represses translation of each of the four target mRNAs via distinct mechanisms. SgrS binding sites overlapping the Shine-Dalgarno sequences of adiY and folE mRNAs suggest that SgrS pairing with these targets directly occludes ribosome binding and prevents translation initiation. SgrS binding within the purR coding sequence recruits the RNA chaperone Hfq to directly repress purR translation. Two separate SgrS binding sites were found on asd mRNA, and both are required for full translational repression. Ectopic overexpression of asd, adiY and folE is specifically detrimental to cells experiencing glucose-phosphate stress, suggesting that SgrS-dependent repression of the metabolic functions encoded by these targets promotes recovery from glucose-phosphate stress. Further studies determined that SgrS regulates its targets with different efficiencies. We showed that SgrS establishes a hierarchy of targets by prioritizing regulation of targets in the following order: 1/2) ptsG and yigL 3) asd 4) manX, 5) purR. However, SgrS binding strength to the target mRNAs is not the sole determinant of regulatory efficiency or prioritization. Looking more carefully at what determines efficiency of SgrS regulation of asd mRNA, we discovered that SgrS binds cooperatively at the two stem structures within asd mRNA. SgrS binding at both sites is not only required for optimal repression of asd translation, but also changes its priority within the regulatory hierarchy. Besides SgrS regulatory mechanisms, this study provides additional insights into the nature of glucose-phosphate stress. Growth experiments in the minimal media demonstrate some differences in toxicity of αMG and 2DG. Importantly, the simultaneous presence of both glucose analogs results in a synthetic phenotype, highly indicative of αMG and 2DG affecting different pathways.
Issue Date:2017-03-10
Type:Thesis
URI:http://hdl.handle.net/2142/97269
Rights Information:Copyright 2017 Maksym Bobrovskyy
Date Available in IDEALS:2017-08-10
Date Deposited:2017-05


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