Riboswitch and small RNAs modulate btuB translation initiation in Escherichia coli and trigger distinct mRNA regulatory mechanisms.

Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.

Cross-ocean patterns and processes in fish biodiversity on coral reefs through the lens of eDNA metabarcoding.

Increasing speed and magnitude of global change threaten the world's biodiversity and particularly coral reef fishes. A better understanding of large-scale patterns and processes on coral reefs is essential to prevent fish biodiversity decline but it requires new monitoring approaches. Here, we use environmental DNA metabarcoding to reconstruct well-known patterns of fish biodiversity on coral reefs and uncover hidden patterns on these highly diverse and threatened ecosystems. We analysed 226 environmental DNA (eDNA) seawater samples from 100 stations in five tropical regions (Caribbean, Central and Southwest Pacific, Coral Triangle and Western Indian Ocean) and compared those to 2047 underwater visual censuses from the Reef Life Survey in 1224 stations. Environmental DNA reveals a higher (16%) fish biodiversity, with 2650 taxa, and 25% more families than underwater visual surveys. By identifying more pelagic, reef-associated and crypto-benthic species, eDNA offers a fresh view on assembly rules across spatial scales. Nevertheless, the reef life survey identified more species than eDNA in 47 shared families, which can be due to incomplete sequence assignment, possibly combined with incomplete detection in the environment, for some species. Combining eDNA metabarcoding and extensive visual census offers novel insights on the spatial organization of the richest marine ecosystems.

Ligand recognition and helical stacking formation are intimately linked in the SAM-I riboswitch regulatory mechanism.

Riboswitches are noncoding mRNA elements that control gene expression by altering their structure upon metabolite binding. Although riboswitch crystal structures provide detailed information about RNA-ligand interactions, little knowledge has been gathered to understand how riboswitches modulate gene expression. Here, we study the molecular recognition mechanism of the S-adenosylmethionine SAM-I riboswitch by characterizing the formation of a helical stacking interaction involving the ligand-binding process. We show that ligand binding is intimately linked to the formation of the helical stacking, which is dependent on the presence of three conserved purine residues that are flanked by stacked helices. We also find that these residues are important for the formation of a crucial long-range base pair formed upon SAM binding. Together, our results lend strong support to a critical role for helical stacking in the folding pathway and suggest a particularly important function in the formation of the long-range base pair.

Role of a hairpin-stabilized pause in the Escherichia coli thiC riboswitch function.

Transcriptional pauses have been reported in bacterial riboswitches and, in some cases, their specific positioning has been shown to be important for gene regulation. Here, we show that a hairpin structure in the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch is involved in transcriptional pausing and ligand sensitivity. Using in vitro transcription kinetic experiments, we show that all three major transcriptional pauses in the thiC riboswitch are affected by NusA, a transcriptional factor known to stimulate hairpin-stabilized pauses. Using a truncated region of the riboswitch, we isolated the hairpin structure responsible for stabilization of the most upstream pause. Destabilization of this structure led to a weaker pause and a decreased NusA effect. In the context of the full-length riboswitch, this same mutation also led to a weaker pause, as well as a decreased TPP binding affinity. Our work suggests that RNA structures involved in transcriptional pausing in riboswitches are important for ligand sensitivity, most likely by increasing the time allowed to the ligand for binding to the riboswitch.

Biophysical Approaches to Bacterial Gene Regulation by Riboswitches.

The last decade has witnessed the discovery of a variety of non-coding RNA sequences that perform a broad range of crucial biological functions. Among these, the ability of certain RNA sequences, so-called riboswitches, has attracted considerable interest. Riboswitches control gene expression in response to the concentration of particular metabolites to which they bind without the need for any protein. These RNA switches not only need to adopt a very specific tridimensional structure to perform their function, but also their sequence has been evolutionary optimized to recognize a particular metabolite with high affinity and selectivity. Thus, riboswitches offer a unique opportunity to get fundamental insights into RNA plasticity and how folding dynamics and ligand recognition mechanisms have been efficiently merged to control gene regulation. Because riboswitch sequences have been mostly found in bacterial organisms controlling the expression of genes associated to the synthesis, degradation or transport of crucial metabolites for bacterial survival, they offer exciting new routes for antibiotic development in an era where bacterial resistance is more than ever challenging conventional drug discovery strategies. Here, we give an overview of the architecture, diversity and regulatory mechanisms employed by riboswitches with particular emphasis on the biophysical methods currently available to characterise their structure and functional dynamics.

Insights into the cotranscriptional and translational control mechanisms of the Escherichia coli tbpA thiamin pyrophosphate riboswitch.

Riboswitches regulate gene expression by modulating their structure upon metabolite binding. These RNA orchestrate several layers of regulation to achieve genetic control. Although Escherichia coli riboswitches modulate translation initiation, several cases have been reported where riboswitches also modulate mRNA levels. Here, we characterize the regulation mechanisms of the thiamin pyrophosphate (TPP) tbpA riboswitch in E. coli. Our results indicate that the tbpA riboswitch modulates both levels of translation and transcription and that TPP sensing is achieved more efficiently cotranscriptionally than post-transcriptionally. The preference for cotranscriptional binding is also observed when monitoring the TPP-dependent inhibition of translation initiation. Using single-molecule approaches, we observe that the aptamer domain freely fluctuates between two main structures involved in TPP recognition. Our results suggest that translation initiation is controlled through the ligand-dependent stabilization of the riboswitch structure. This study demonstrates that riboswitch cotranscriptional sensing is the primary determinant in controlling translation and mRNA levels.