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RNAs are often studied in nonnative sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods—a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added postprobing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5′ and 3′ ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer-only construct but introduced nonnative interactions in the full-length riboswitch. In contrast, Switch-MaP provided reactivity data for all nucleotides in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is a straightforward alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to nonnative sequence elements. 

SreA is one of seven candidate S-adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes, a saprophyte and opportunistic foodborne pathogen. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine, and is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenes persists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a similar structure and SAM binding properties to other well characterized SAM-I riboswitches. Despite apparent structural similarities to previously described SAM-I riboswitches, SreA induces transcription termination in response to comparatively lower (nM) ligand concentrations. Furthermore, SreA is a leaky riboswitch that permits some transcription of the downstream even in the presence of mM SAM suggesting that L. monocytogenes may “dampen” the expression of genes for methionine import, but likely does not turn them “OFF”. 

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. 

The mid-Pleistocene transition (MPT) [~1.25 to 0.85 million years ago (Ma)] marks a shift in the character of glacial-interglacial climate. One prevailing hypothesis for the origin of the MPT is that glacial deep ocean circulation fundamentally changed, marked by a circulation “crisis” at ~0.90 Ma (marine isotope stages 24 to 22). Using high-resolution paired neodymium, carbon, and oxygen isotope data from the South Atlantic Ocean (Cape Basin) across the MPT, we find no evidence of a substantial change in deep ocean circulation. Before and during the early MPT (~1.30 to 1.12 Ma), the glacial deep ocean variability closely resembled that of the most recent glacial cycle. The carbon storage facilitated by developing deep ocean stratification across the MPT required only modest circulation adjustments. 

Abstract In the southern Indian Ocean, the position of the subtropical front – the boundary between colder, fresher waters to the south and warmer, saltier waters to the north – has a strong influence on the upper ocean hydrodynamics and biogeochemistry. Here we analyse a sedimentary record from the Agulhas Plateau, located close to the modern position of the subtropical front and use alkenones and coccolith assemblages to reconstruct oceanographic conditions over the past 300,000 years. We identify a strong glacial-interglacial variability in sea surface temperature and productivity associated with subtropical front migration over the Agulhas Plateau, as well as shorter-term high frequency variability aligned with variations in high latitude insolation. Alkenone and coccolith abundances, in combination with diatom and organic carbon records indicate high glacial export productivity. We conclude that the biological pump was more efficient and strengthened during glacial periods, which could partly account for the reported reduction in atmospheric carbon dioxide concentrations.