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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”. 

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. 

Abstract A common conception of the deep ocean during ice age episodes is that the upper circulation cell in the Atlantic was shoaled at the Last Glacial Maximum compared to today, and that this configuration facilitated enhanced carbon storage in the deep ocean, contributing to glacial CO₂draw‐down. Here, we test this notion in the far South Atlantic, investigating changes in glacial circulation structure using paired neodymium and benthic carbon isotope measurements from International Ocean Discovery Program Site U1479, at 2,615 m water depth in the Cape Basin. We infer changes in circulation structure across the last glacial cycle by aligning our site with other existing carbon and neodymium isotope records from the Cape Basin, examining vertical isotope gradients, while determining the relative timing of inferred circulation changes at different depths. We find that Site U1479 had the most negative neodymium isotopic composition across the last glacial cycle among the analyzed sites, indicating that this depth was most strongly influenced by North Atlantic Deep Water (NADW) in both interglacial and glacial intervals. This observation precludes a hypothesized dramatic shoaling of NADW above ∼2,000 m. Our evidence, however, indicates greater stratification between mid‐depth and abyssal sites throughout the last glacial cycle, conditions that developed in Marine Isotope Stage 5. These conditions still may have contributed to glacial carbon storage in the deep ocean, despite little change in the mid‐depth ocean structure. 

Abstract The meridional variability of the Subtropical Front (STF) in the Southern Hemisphere, linked to expansions or contractions of the Southern Ocean, may have played an important role in global ocean circulation by moderating the magnitude of water exchange at the Indian‐Atlantic Ocean Gateway, so called Agulhas Leakage. Here we present new biomarker records of upper water column temperature (and) and primary productivity (chlorins and alkenones) from marine sediments at IODP Site U1475 on the Agulhas Plateau, near the STF and within the Agulhas retroflection pathway. We use these multiproxy time‐series records from 1.4 to 0.3 Ma to examine implied changes in the upper oceanographic conditions at the mid‐Pleistocene transition (MPT, ca. 1.2–0.8 Ma). Our reconstructions, combined with prior evidence of migrations of the STF over the last 350 ka, suggest that in the Southwestern Indian Ocean the STF may have been further south from the Agulhas Plateau during the mid‐Pleistocene Interim State (MPIS, MIS 23–12) and reached its northernmost position during MIS 34–24 and MIS 10. Comparison to aGloborotalia menardii‐derived Agulhas Leakage reconstruction from the Cape Basin suggests that only the most extreme northward migrations of the STF are associated with reduced Agulhas Leakage. During the MPIS, STF migrations do not appear to control Agulhas Leakage variability, we suggest previously modeled shifting westerly winds may be responsible for the patterns observed. A detachment between STF migrations and Agulhas Leakage, in addition to invoking shifting westerly winds may also help explain changes in CO₂ventilation seen during the MPIS.