Characterization of non-coding regulatory RNA from Listeria monocytogenes

Abstract

Non-coding (nc) RNAs enable rapid and adaptable control of gene expression. For example, micro (mi) RNAs, piwi-interacting (pi) RNAs, and small interfering (si) RNAs regulate gene expression post-transcriptionally by base pairing complementary targets. However, some ncRNAs adopt complex structures that change in response to environmental stimuli like temperature, pH, and concentrations of ions or other molecules. In the appropriate genomic context, these conformationally flexible ncRNAs can control gene expression. Two classes of structured ncRNAs enable the opportunistic human pathogen Listeria monocytogenes to adapt to the host environment and cause listeriosis. An RNA thermosensor (RNAT) in the 5′ untranslated region (UTR) of the prfA messenger RNA controls the translation of positive regulatory factor A (PrfA), a transcription factor that activates the expression of key virulence genes, in a temperature-dependent manner. At low temperatures, the ribosome binding site (RBS) is occluded from the 30S ribosome repressing translation initiation. At elevated temperatures, the RBS becomes accessible, permitting translation initiation. This temperature-sensitive mechanism ensures that L. monocytogenes expresses virulence genes specifically upon host invasion. S-adenosylmethionine (SAM) class I riboswitches are a second family of regulatory RNAs in L. monocytogenes. SAM-I riboswitches form intrinsic terminator hairpins in a SAM-dependent manner that cause premature Rho-independent transcription termination of various sulfur metabolism genes. SAM riboswitch elements (SreA - G) were identified as candidate SAM-I riboswitches in L. monocytogenes, yet had not been functionally validated. In this thesis, I investigated how changes in the structures of the prfA RNAT and SreA - E control gene expression in L. monocytogenes. The energetics underlying the temperature- or ligand-dependent conformational changes were studied by circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). We examined the structural changes of the RNAs at the base pair level by nuclear magnetic resonance (NMR) spectroscopy and chemical probing, whereas global RNA structures were monitored by small angle X-ray scattering (SAXS). To relate changes in RNA structure to gene expression under varied conditions, we used transcription and translation assays. I found that the stability of the prfA RNAT global structure controls translation of the downstream gene and that stabilizing regions distal to the RBS reduced translation to a similar extent as stabilizing RBS proximal structures. Additionally, and unlike simpler hairpin RNATs, the unfolding of the prfA RNAT proceeds through a two-transition model where internal loops modulate unfolding cooperativity. These findings challenge the axiom that only local structure near the RBS is important for controlling translation of the downstream gene and diversify the known mechanisms of RNAT unfolding and translational control. For the candidate SAM-1 riboswitches, I validated ligand binding and ligand induced conformational changes that are required for transcriptional control. SreA - C share the highly conserved SAM-I riboswitch sequence yet control the transcription of sulfur metabolism genes in a SAM-dependent manner with distinct sensitivities to the rate of transcription. Despite apparent sequence similarities, the co-transcriptional folding of SreA - C tailor transcription termination to meet the distinct regulatory requirements of the downstream genes. These findings suggest that sequence variation in strand displacement regions may modulate transcription termination. Collectively, the present work informs on how L. monocytogenes uses structured ncRNAs to control gene expression required for its biphasic lifestyle. Furthermore, this detailed characterization of ncRNA elements supports future multidisciplinary research to develop strategies for the prevention or treatment of listeriosis infections.