Poster abstracts

Poster number 6 submitted by Jocelyn Chen

A regulatory RNA:RNA interaction between a SAM-I riboswitch and RNA thermosensor in Listeria monocytogenes

Jocelyn V. Chen (Department of Chemistry, University of Michigan), Martin OSteen (Program in Biophysics, University of Michigan), Antigone N. Wilson (Department of Chemistry, University of Michigan), Aldrex Munsayac (Department of Chemistry, University of Michigan), Sarah C. Keane (Department of Chemistry and Program in Biophysics, University of Michigan)

Abstract:
Regulatory non-coding (nc) RNAs are RNA elements that function to control expression of their downstream genes. In Listeria monocytogenes, a pathogenic bacterium that causes deadly foodborne illnesses, ncRNAs play an important role in regulating the expression of its virulence genes. The master regulator of the virulence genes in L. monocytogenes is the transcription factor Positive Regulatory Factor A (PrfA). The translation of PrfA is controlled by the prfA RNA thermosensor (RNAT), a structured ncRNA found in the 5’ untranslated region (UTR) of the prfA mRNA, which unfolds to promote translation at elevated temperatures such as inside a human host. Additionally, the S-adenosylmethionine (SAM) riboswitch element A (SreA) is another structured ncRNA found in the 5’ UTR that functions by sensing and binding to its cognate ligand and metabolite, SAM, which results in a conformational change that terminates transcription of its downstream genes that encode a methionine ABC transporter. Although the prfA RNAT and the SreA riboswitch can individually function in cis to regulate their downstream genes, previous studies have shown that SreA can form an RNA:RNA complex with prfA and interact in trans to inhibit PrfA expression. However, this interaction remains largely uncharacterized at the mechanistic level. To better understand the RNA:RNA interaction between the prfA RNAT and the SreA riboswitch, we designed a library of SreA mutant constructs to identify regions at the molecular level that are critical for the prfA:SreA interaction. We also performed electrophoretic mobility shift assays (EMSAs) and in vitro transcription-translation assays to assess sequence dependence of each helical region and validate translational inhibition of the PrfA protein. These studies will provide insights into how ncRNAs in L. monocytogenes regulate gene expression and control virulence.

Keywords: Riboswitch, RNA thermosensor, non-coding RNA