The Cellular Environment Stabilizes Adenine Riboswitch RNA Structure

Tyrrell, Jillian; McGinnis, Jennifer L.; Weeks, Kevin M.; Pielak, Gary J.

doi:10.1021/bi401207q

Cited by 108 publications

(167 citation statements)

References 60 publications

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“…An advantage of SHAPE probing compared with DMS probing is that it provides information for all positions rather than only the adenine and cytosine positions and for in vitro RNA structure-probing experiments SHAPE has been the preferred method. The development of new SHAPE reagents based on imidazolide chemistry, which can probe RNA structure inside living cells (Spitale et al 2013), and the adaption of well-known SHAPE reagents to in vivo probing (Tyrrell et al 2013) suggest that SHAPE methodology eventually can be applied to global probing of in vivo RNA structure. However, it is clear that global in vivo SHAPE probing will be more challenging than the probing of single RNA molecules in vitro.…”

Section: Shape Reagents React With Thementioning

confidence: 99%

SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

Poulsen

Kielpinski

Salama

et al. 2015

RNA

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Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA-RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing.

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Section: Shape Reagents React With Thementioning

confidence: 99%

SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

Poulsen

Kielpinski

Salama

et al. 2015

RNA

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“…Ligand binding was tested under physiological monovalent cation concentrations (135 and 15 mM NaCl) and 10 mM magnesium chloride, a divalent cation concentration that promotes RNA folding in vitro to a similar extent as observed in vivo for the purine riboswitch aptamer domain (18). Wild-type and a minimized aptamer containing truncations in P2 and P4 [env87(minimal)] display ∼100 nM affinity for SAM (Table 1, Table S2, and Fig.…”

Section: Rna Crystallization and Structure Determinationmentioning

confidence: 99%

Structural basis for diversity in the SAM clan of riboswitches

Trausch¹,

Xu²,

Edwards³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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In bacteria, sulfur metabolism is regulated in part by seven known families of riboswitches that bind S-adenosyl-L-methionine (SAM). Direct binding of SAM to these mRNA regulatory elements governs a downstream secondary structural switch that communicates with the transcriptional and/or translational expression machinery. The most widely distributed SAM-binding riboswitches belong to the SAM clan, comprising three families that share a common SAMbinding core but differ radically in their peripheral architecture. Although the structure of the SAM-I member of this clan has been extensively studied, how the alternative peripheral architecture of the other families supports the common SAM-binding core remains unknown. We have therefore solved the X-ray structure of a member of the SAM-I/IV family containing the alternative "PK-2" subdomain shared with the SAM-IV family. This structure reveals that this subdomain forms extensive interactions with the helix housing the SAM-binding pocket, including a highly unusual mode of helix packing in which two helices pack in a perpendicular fashion. Biochemical and genetic analysis of this RNA reveals that SAM binding induces many of these interactions, including stabilization of a pseudoknot that is part of the regulatory switch. Despite strong structural similarity between the cores of SAM-I and SAM-I/IV members, a phylogenetic analysis of sequences does not indicate that they derive from a common ancestor.RNA structure | X-ray crystallography | chemical probing | isothermal titration calorimetry | gene regulation R iboswitches are noncoding RNA elements generally found in the leader of bacterial mRNAs that regulate expression via direct binding of a specific cellular metabolite (reviewed in refs.

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“…1B; Watters et al 2016). In this measurement, SHAPE reagents introduced in cell cultures modify cellular RNAs at positions that are unstructured (Tyrrell et al 2013;McGinnis and Weeks 2014). After RNA extraction, modification positions are identified through reverse transcription, which is blocked by the modification.…”

Section: Introductionmentioning

confidence: 99%

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

Takahashi

Watters

Gasper

et al. 2016

RNA

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Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure-function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure-function design principles for a diverse array of natural and synthetic RNA regulators.

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The Cellular Environment Stabilizes Adenine Riboswitch RNA Structure

Cited by 108 publications

References 60 publications

SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

Structural basis for diversity in the SAM clan of riboswitches

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

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