Poster abstracts

Poster number 44 submitted by Ian Hall

Characterization of putative SAM-I riboswitches of L. monocytogenes

Ian Hall (Department of Chemistry, University of Michigan), Sarah Keane (Department of Chemistry and Program in Biophysics, University of Michigan)

Abstract:
Listeria monocytogenes is a saprophytic bacterium which can infect humans causing listeriosis, a life-threatening foodborne illness. In the U.S. about 2,500 cases of listeriosis and 500 related deaths are reported each year. Understanding the bacteria’s virulence and metabolism are essential for improving strategies to prevent and treat listeriosis. Notably regulation of the bacteria’s virulence gene expression is primarily controlled by positive regulatory factor A (PrfA) and an RNA thermosensor (RNAT) that resides in the 5′ untranslated region (UTR) of the prfA messenger (m)RNA. We previously described the mechanism of the prfA RNAT’s temperature dependent translation regulation.1 PrfA translation research revealed a novel trans interaction between the prfA RNAT and S-adenosyl methionine (SAM) response element A (SreA)2, a putative SAM-I riboswitch identified in tiling arrays of the L. monocytogenes genome.3
The present work biophysically characterizes SreA and six other putative SAM-I riboswitches of L. monocytogenes (SreA-G). These elements reside in front of genes for SAM biosynthesis pathways and/or SAM related transport pathways. A key hypothesis of this work is whether the genomic location of a riboswitch is related to its regulatory strength. To answer this question and characterize these presumed SAM-I riboswitches, we initiated studies on the ligand binding, transcription termination, and structure of SreA-G. The ligand affinity and specificity of SreA-G are being determined by isothermal titration calorimetry (ITC) using cognate (SAM) and near-cognate (SAH) ligands. The transcription termination of the riboswitches with SAM or SAH are being evaluated by a 32P GTP body labeled assay. The aptamer domains of the riboswitches were studied by small angle X-ray scattering (SAXS) with and without SAM, and in all cases, we observe compaction of the structure with SAM. Additional structural studies of aptamer domains via X-ray crystallography are underway.

References:
1. Zhang, H.; Hall, I.; Nissley, A. J.; Abdallah, K.; Keane, S. C., A Tale of Two Transitions: The Unfolding Mechanism of the prfA RNA Thermosensor. Biochemistry 2020, 59 (48), 4533-4545.
2. Loh, E.; Dussurget, O.; Gripenland, J.; Vaitkevicius, K.; Tiensuu, T.; Mandin, P.; Repoila, F.; Buchrieser, C.; Cossart, P.; Johansson, J., A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes. Cell 2009, 139 (4), 770-9.
3. Toledo-Arana, A.; Dussurget, O.; Nikitas, G.; Sesto, N.; Guet-Revillet, H.; Balestrino, D.; Loh, E.; Gripenland, J.; Tiensuu, T.; Vaitkevicius, K.; Barthelemy, M.; Vergassola, M.; Nahori, M.-A.; Soubigou, G.; Régnault, B.; Coppée, J.-Y.; Lecuit, M.; Johansson, J.; Cossart, P., The Listeria transcriptional landscape from saprophytism to virulence. Nature 2009, 459 (7249), 950-956.

Keywords: SAM-I riboswitch, Listeria monocytogenes