Heterogeneous ribosomes and bS21: An investigation of impacts and mechanisms of altered translation in Francisella tularensis

Author

Hannah Trautmann, University of Rhode IslandFollow

Date of Award

1-1-2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological and Environmental Sciences

Specialization

Cell and Molecular Biology

Department

Cell & Molecular Biology

First Advisor

Kathryn Ramsey

Abstract

Francisella tularensis is a pathogenic bacterium that requires coordinated regulation of gene expression to cause disease. In this dissertation, I explore a novel area of translation-level gene regulation in F. tularensis: heterogeneous ribosomes that preferentially translate specific mRNAs. Through mass spectrometry and immunoblotting, I find that wild-type F. tularensis ribosomes are heterogeneous with respect to the small ribosomal protein bS21 and all three bS21 homologs are able to be incorporated into ribosomes. Comparing proteomics and transcriptomics data, I determine that cells lacking one homolog, bS21-2, have genome-wide changes in protein abundance that are not explained by changes in transcript abundance. Among the impacted genes are key virulence factors encoding the Type VI Secretion System. I also show that cells lacking bS21-2 are defective in intramacrophage replication.

I then explore the mechanism that causes these changes in gene expression using reporter fusion assays. I determine that the 5´ untranslated regions (UTRs) of some genes are sufficient to drive differences in reporter protein abundance, independently of the RNA chaperone protein Hfq. While I do not find conserved structural motifs in bS21-2-responsive 5´ UTRs, I identify a 6-nucleotide sequence in the 5´ UTR of mraY that is necessary to cause differences in translation when bS21-2 is absent. I also determine that genes with perfect Shine-Dalgarno sequences do not require bS21-2 for efficient translation. Together, these findings strongly suggest that F. tularensis contains heterogeneous ribosomes that differentially translate mRNA molecules with specific leader sequences, causing downstream impacts on the type VI secretion system and virulence.

Recommended Citation

Trautmann, Hannah, "Heterogeneous ribosomes and bS21: An investigation of impacts and mechanisms of altered translation in Francisella tularensis" (2023). Open Access Dissertations. Paper 1539.
https://digitalcommons.uri.edu/oa_diss/1539