Talk Abstracts

Talk on Friday 01:15-01:30pm submitted by David Beier

Linking local RNA structural dynamics to ribosome binding in the prfA RNA thermosensor

David Beier (Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA), Elizabeth Duran (Biophysics Program, University of Michigan, Ann Arbor, Michigan 48109, USA), Nils Walter (Department of Biological Chemistry and Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA), Sarah Keane (Biophysics Program and Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA)

Abstract:
The 5′-untranslated region (UTR) of the Listeria monocytogenes prfA messenger (m) RNA controls the translation of the PrfA transcriptional regulator in a temperature dependent manner¹. PrfA is an important transcription factor that controls the expression of a cluster of virulence genes involved in pathogenesis². At low temperature (4-30 ^oC) the prfA mRNA transcript is detected, however translation of PrfA does not occur. Concurrent with a temperature shift to 37 ^oC upon successful infection of a human host, translation of PrfA occurs. Since mRNA levels of prfA remain similar as protein levels change, the 5′-UTR of prfA is characterized as an RNA thermosensor (RNAT). Thorough analysis of the prfA 5′-UTR is essential to better understand how RNA-mediated processes in L. monocytogenes initiate and prolong infection.

The Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) assay uses transient fluorescent oligonucleotides to probe the accessibility of specific regions within an RNA structure³. Monitoring binding events to a specific region of an RNA allows for the determination of RNA structure accessibility, and ultimately the conformational dynamics of the RNA. This analysis can be used to identify regions of the prfA 5′-UTR that unfold with increasing temperature to expose elements required for translation initiation. Our SiM-KARTS data reveals that the initial destabilization of the local RNA structure occurs between 30 and 34 ^oC. This destabilization occurs proximal to the apical loop of the RNAT, upstream of the Shine-Dalgarno (SD) sequence. Unfolding near the SD sequence occurs between 34 and 37 ^oC. We do not observe destabilization of the helical region containing the AUG start codon at the temperatures probed. Fluorescent ribosomal toeprinting suggests that the interaction of the prfA RNAT and 70S ribosome is temperature dependent, as truncated cDNA products are first observed at 37 ^oC, but not 30 or 34 ^oC. Together, these results indicate that the stability of the local structure at and upstream of the SD sequence, but not the AUG start codon, is essential for ribosome binding.

References:
(1) Johansson, J.; Mandin, P.; Renzoni, A.; Chiaruttini, C.; Springer, M.; Cossart, P. An RNA Thermosensor Controls Expression of Virulence Genes in Listeria Monocytogenes. Cell 2002, 110 (5), 551–561. https://doi.org/10.1016/S0092-8674(02)00905-4.
(2) 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. https://doi.org/10.1021/ACS.BIOCHEM.0C00588/SUPPL_FILE/BI0C00588_SI_001.PDF.
(3) Chauvier, A.; Cabello-Villegas, J.; Walter, N. G. Probing RNA Structure and Interaction Dynamics at the Single Molecule Level. Methods 2019, 162–163, 3–11. https://doi.org/10.1016/J.YMETH.2019.04.002.

Keywords: Thermosensor, Single-Molecule, Structural Dynamics