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The cellular environment stabilizes adenine riboswitch RNA structure.

ABSTRACT: There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute time scale using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension). The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg(2+), the approximate Mg(2+) concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg(2+) concentrations to the buffer used for in vitro analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg(2+) alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations.

SUBMITTER: Tyrrell J

PROVIDER: S-EPMC4905562 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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The cellular environment stabilizes adenine riboswitch RNA structure.

Tyrrell Jillian J McGinnis Jennifer L JL Weeks Kevin M KM Pielak Gary J GJ

Biochemistry 20131120 48

There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute time scale using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension). The ligand-bound apt ...[more]

PMID: 24215455

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Project description:Expression of a large fraction of genes in bacteria is controlled by riboswitches, which are found in the untranslated region of mRNA. Structurally riboswitches have a conserved aptamer domain to which a metabolite binds, resulting in a conformational change in the downstream expression platform. Prediction of the functions of riboswitches requires a quantitative description of the folding landscape so that the barriers and time scales for the conformational change in the switching region in the aptamer can be estimated. Using a combination of all atom molecular dynamics (MD) and coarse-grained model simulations we studied the response of adenine (A) binding add and pbuE A-riboswitches to mechanical force. The two riboswitches contain a structurally similar three-way junction formed by three paired helices, P1, P2, and P3, but carry out different functions. Using pulling simulations, with structures generated in MD simulations, we show that after P1 rips the dominant unfolding pathway in the add A-riboswitch is the rupture of P2 followed by unraveling of P3. In the pbuE A-riboswitch, after P1 unfolds P3 ruptures ahead of P2. The order of unfolding of the helices, which is in accord with single molecule pulling experiments, is determined by the relative stabilities of the individual helices. Our results show that the stability of isolated helices determines the order of assembly and response to force in these non-coding regions. We use the simulated free energy profile for the pbuE A-riboswitch to estimate the time scale for allosteric switching, which shows that this riboswitch is under kinetic control lending additional support to the conclusion based on single molecule pulling experiments. A consequence of the stability hypothesis is that a single point mutation (U28C) in the P2 helix of the add A-riboswitch, which increases the stability of P2, would make the folding landscapes of the two riboswitches similar. This prediction can be tested in single molecule pulling experiments.

Project description:INTRODUCTION: Riboswitches are cis-acting regulatory RNA elements prevalently located in the leader sequences of bacterial mRNA. An adenine sensing riboswitch cis-regulates adeninosine deaminase gene (add) in Vibrio vulnificus. The structural mechanism regulating its conformational changes upon ligand binding mostly remains to be elucidated. In this open framework it has been suggested that the ligand stabilizes the interaction of the distal "kissing loop" complex. Using accurate full-atom molecular dynamics with explicit solvent in combination with enhanced sampling techniques and advanced analysis methods it could be possible to provide a more detailed perspective on the formation of these tertiary contacts. METHODS: In this work, we used umbrella sampling simulations to study the thermodynamics of the kissing loop complex in the presence and in the absence of the cognate ligand. We enforced the breaking/formation of the loop-loop interaction restraining the distance between the two loops. We also assessed the convergence of the results by using two alternative initialization protocols. A structural analysis was performed using a novel approach to analyze base contacts. RESULTS: Contacts between the two loops were progressively lost when larger inter-loop distances were enforced. Inter-loop Watson-Crick contacts survived at larger separation when compared with non-canonical pairing and stacking interactions. Intra-loop stacking contacts remained formed upon loop undocking. Our simulations qualitatively indicated that the ligand could stabilize the kissing loop complex. We also compared with previously published simulation studies. DISCUSSION AND CONCLUSIONS: Kissing complex stabilization given by the ligand was compatible with available experimental data. However, the dependence of its value on the initialization protocol of the umbrella sampling simulations posed some questions on the quantitative interpretation of the results and called for better converged enhanced sampling simulations.

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The cellular environment stabilizes adenine riboswitch RNA structure.

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The cellular environment stabilizes adenine riboswitch RNA structure.

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