Semester

Fall

Date of Graduation

2009

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Thomas Elliott.

Abstract

The first part of this thesis is dedicated to translational regulation of rpoS mRNA by the small noncoding RNAs (sRNAs), DsrA and RprA, in two closely related enteric bacteria, Escherichia coli, and Salmonella enterica serovar Typhimurium. The rpoS gene encodes a second vegetative sigma factor for RNA polymerase, which directs the cell's transcriptional response to general stress and entry into stationary phase. The rpoS gene is highly conserved among the gamma-branch of proteobacteria, and sRNAs are highly conserved in related species. In fact, sequence conservation is thought to have predictive value in sRNA discovery and functional conservation is largely assumed. First discovered in E. coli, DsrA and RprA were shown to activate rpoS translation in response to low temperature and osmotic shock respectively. Base pairing between these sRNAs and rpoS mRNA disrupts a hairpin in the untranslated leader region of rpoS that blocks ribosome binding. The function of these sRNAs was tested in S. enterica serovar Typhimurium under the same conditions reported to be important for their function in E. coli . Neither DsrA nor RprA was required for rpoS regulation in S. enterica. Importantly, this work demonstrates that sRNA function cannot be inferred from sequence conservation.;The second part of this thesis provides evidence for a model in which heme biosynthesis in S. enterica is feedback regulated by heme at HemA, the enzyme catalyzing the first committed step of the pathway. HemA is primarily regulated by conditional stability, becoming more stable in response to heme limitation and subject to rapid turnover by ClpAP and Lon proteases when not limited for heme. The first 18 amino acids of HemA are sufficient for protease recognition, however other regions of the protein are required for heme-responsiveness. Although examples of direct feedback inhibition by heme exist in other organisms, the mechanism by which HemA is targeted for proteolysis in S. enterica is unknown. A model in which heme functions as a proteolytic tag by directly binding HemA is supported by the following: (i) Purified HemA from S. enterica contains bound heme, (ii) mutation of a single cysteine residue (C170) results in purified HemA that lacks bound heme, and (iii) the C170A mutant protein is stable in vivo.

Download

Share

COinS