Unprecedented tunability of riboswitch structure and regulatory function by sub-millimolar variations in physiological Mg2+

Abstract

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Transcriptional Riboswitches Integrate Timescales for Bacterial Gene Expression Control

“…This dependence of the folding pathway on the relative timescales of ligand binding and conformational dynamics of the aptamer can be identified as a kinetic coupling mechanism occurring early in the decision tree of gene regulation ( Figure 1B ). Similar kinetic control mechanisms of ligand recognition by the aptamer have been observed to be operational in multiple other riboswitches (Manz et al, 2017 ; Rode et al, 2018 ; McCluskey et al, 2019 ; Sung and Nesbitt, 2019 ).…”

Combining Coarse-Grained Simulations and Single Molecule Analysis Reveals a Three-State Folding Model of the Guanidine-II Riboswitch

“…With regard to the Mg 2+ titration, a consistent picture emerges from our combined approach of smFRET experiments and coarse-grained simulations. While smFRET experiments are routinely employed to characterize the Mg 2+ -dependent folding of RNAs ( Lambert et al, 2005 ; Steiner et al, 2008 ; Kobitski et al, 2011 ; Hengesbach et al, 2012 ; Bisaria et al, 2016 ) and in particular riboswitches ( Manz et al, 2017 ; Warhaut et al, 2017 ; McCluskey et al, 2019 ; Warnasooriya et al, 2019 ), the combined approach used here allows us to provide a more comprehensive view. The quantitative agreement between the maximum efficiencies of each state from simulations and experiments highlights the compatibility of both approaches despite possible limitations in the time scale accessible to the simulations.…”

An NMR-based approach reveals the core structure of the functional domain of SINEUP lncRNAs

“…Secondary structure determination of such structured RNA domains is even more crucial than that of proteins, because RNA is capable not only of folding in multiple, different structures (which are often not functional), but also of folding by using artificial base-pairings that are not formed in native RNA. For instance, several functional RNAs can adopt two structures, such as riboswitches ( 51 ). The 1D 1 H NMR spectra of the imino proton region were useful in our initial analysis of domain structure.…”

“…An understanding of the structures of lncRNAs is a crucial step toward understanding their cellular mechanisms. However, challenges with in vitro NMR structural analysis still remain, because RNAs adapt to different regulatory states by structural change across different cellular compartments ( 51 ). Because AS-Uchl1 RNAs and synthetic SINEUPs are intercellular mobile elements, nucleocytoplasmic shuttling happens after transcription ( 10 , 55 ).…”