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Observation of preQ1-II riboswitch dynamics using single-molecule FRET.

ABSTRACT: PreQ1 riboswitches regulate the synthesis of the hypermodified tRNA base queuosine by sensing the pyrrolopyrimidine metabolite preQ1. Here, we use single-molecule FRET to interrogate the structural dynamics of apo and preQ1-bound states of the preQ1-II riboswitch from Lactobacillus rhamnosus. We find that the apo-form of the riboswitch spontaneously samples multiple conformations. Magnesium ions and preQ1 stabilize conformations that sequester the ribosome-binding site of the mRNA within the pseudoknotted structure, thus inhibiting translation initiation. Our results reveal that folding of the preQ1-II riboswitch is complex and provide evidence favoring a conformational selection model of effector binding by riboswitches of this class.

SUBMITTER: Warnasooriya C

PROVIDER: S-EPMC6693549 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Observation of preQ1-II riboswitch dynamics using single-molecule FRET.

Warnasooriya Chandani C Ling Clarence C Belashov Ivan A IA Salim Mohammad M Wedekind Joseph E JE Ermolenko Dmitri N DN

RNA biology 20181030 9

PreQ1 riboswitches regulate the synthesis of the hypermodified tRNA base queuosine by sensing the pyrrolopyrimidine metabolite preQ1. Here, we use single-molecule FRET to interrogate the structural dynamics of apo and preQ1-bound states of the preQ1-II riboswitch from Lactobacillus rhamnosus. We find that the apo-form of the riboswitch spontaneously samples multiple conformations. Magnesium ions and preQ1 stabilize conformations that ...[more]

PMID: 30328747

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Project description:Riboswitches represent a family of highly structured regulatory elements found primarily in the leader sequences of bacterial mRNAs. They function as molecular switches capable of altering gene expression; commonly, this occurs via a conformational change in a regulatory element of a riboswitch that results from ligand binding in the aptamer domain. Numerous studies have investigated the ligand binding process, but little is known about the structural changes in the regulatory element. A mechanistic description of both processes is essential for deeply understanding how riboswitches modulate gene expression. This task is greatly facilitated by studying all aspects of riboswitch structure/dynamics/function in the same model system. To this end, single-molecule fluorescence resonance energy transfer (smFRET) techniques have been used to directly observe the conformational dynamics of a hydroxocobalamin (HyCbl) binding riboswitch (env8HyCbl) with a known crystallographic structure.1 The single-molecule RNA construct studied in this work is unique in that it contains all of the structural elements both necessary and sufficient for regulation of gene expression in a biological context. The results of this investigation reveal that the undocking rate constant associated with the disruption of a long-range kissing-loop (KL) interaction is substantially decreased when the ligand is bound to the RNA, resulting in a preferential stabilization of the docked conformation. Notably, the formation of this tertiary KL interaction directly sequesters the Shine-Dalgarno sequence (i.e., the ribosome binding site) via base-pairing, thus preventing translation initiation. These results reveal that the conformational dynamics of this regulatory switch are quantitatively described by a four-state kinetic model, whereby ligand binding promotes formation of the KL interaction. The results of complementary cell-based gene expression experiments conducted in Escherichia coli are highly correlated with the smFRET results, suggesting that KL formation is directly responsible for regulating gene expression.

Dataset Information

Observation of preQ1-II riboswitch dynamics using single-molecule FRET.

Publications

Observation of preQ<sub>1</sub>-II riboswitch dynamics using single-molecule FRET.

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