Mechanistic and physiological insights into post-transcriptional regulation by small RNAs SgrS and DicF

Balasubramanian, Divya

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Description

Title

Mechanistic and physiological insights into post-transcriptional regulation by small RNAs SgrS and DicF

Author(s)

Balasubramanian, Divya

Issue Date

2014-09-16

Director of Research (if dissertation) or Advisor (if thesis)

Vanderpool, Carin K.

Doctoral Committee Chair(s)

Vanderpool, Carin K.

Committee Member(s)

• Gardner, Jeffrey F.
• Metcalf, William W.
• Slauch, James M.

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• small RNAs
• SgrS
• DicF
• Mechanisms of regulation

Abstract

Base pairing small regulatory RNAs (sRNAs) are important post-transcriptional regulators of gene expression in bacteria. These sRNAs deploy novel mechanisms to regulate mRNA targets leading to various physiological outcomes during stress conditions including, but not limited to, iron starvation, carbon flux and metabolism, virulence, and quorum sensing. In this study, we investigate the multitude of clever mechanisms that two sRNAs, SgrS and DicF, utilize to regulate gene expression, and the physiological consequences of such regulation. The SgrS sRNA participates in a response to a growth inhibitory stress condition called sugar-phosphate stress caused by the toxic accumulation of phosphorylated sugars. SgrS combats this stress with its RNA base pairing function by silencing translation of sugar transporters that import the stress molecules. SgrS was previously shown to negatively regulate the manXYZ broad sugar-substrate transporter. In this study we demonstrate that SgrS binds at manX and in the intergenic region of manXY to translationally silence this operon. We show that pairing at both these sites is critical for degradation of the manXYZ polycistron, and is also crucial for providing maximal relief from stress. SgrS is a dual-function sRNA in that in addition to its RNA function, it also produces a peptide called SgrT. Here, we investigate the mechanistic relationship between these two functions of SgrS. We demonstrate that while mutating the sgrT translation initiation sequences have little impact on SgrS base pairing properties, mutations in the sgrS base pairing region increase SgrT production. Further, the SgrS RNA function was the primary means of action against sugar-phosphate stress; SgrT production lags SgrS synthesis. We therefore propose a model in which the two independent functions of SgrS act at different stages in response to sugar-phosphate stress. The DicF sRNA is encoded on the dicBF operon on the cryptic Qin prophage in E. coli. The only known target of DicF is the ftsZ mRNA, whose gene product is essential for cell division in bacteria. Using reporter gene fusions to predicted target genes, we identified three genes in metabolism that are also targets of DicF. DicF also requires core E. coli proteins for its base pairing properties. We also demonstrate that in addition to inhibiting cell division, DicF leads to growth inhibition of cells. Lastly, the gene products of the dicBF operon are cumulatively toxic to E. coli cells. Characterizing mechanistic contributions of sRNAs to significant physiological outcomes will better our understanding of these novel regulatory RNA elements.

Graduation Semester

2014-08

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Copyright 2014 Divya Balasubramanian

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