Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold

Liberman, Joseph A.; Salim, Mohammad; Krucinska, J.; Wedekind, Joseph E.

doi:10.1038/nchembio.1231

Cited by 79 publications

(140 citation statements)

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“…S1A; Methods and SI Methods). These results are comparable to values measured for class I and II family members, which produced apparent K D values of 7.3 nM and 17.9 nM, respectively, and showed similar 1:1 binding stoichiometry (11,18). Crystals of the 101-mer were grown from organic salts at neutral pH and the phase problem was overcome by single-isomorphous replacement with anomalous scattering; the initial structure was then refined to 2.75 Å resolution to acceptable R factor and geometry values (Table 1).…”

Section: Resultssupporting

confidence: 75%

“…Frequent docking of the RBS would sequester the expression platform, leading to ligand-dependent queT gene control by translational attenuation. This paradigm differs from other riboswitches, such as preQ 1 -II and SAM-II, because these molecules integrate RBS sequences directly into their aptamer domains upon ligand binding (8,10,12,16,18). Consequently, RBS docking within these riboswitches is characterized by prolonged high-FRET dwell times in the presence of Mg 2+ and ligand (>2.2 s and ∼3.5 s, respectively) with timescales limited most likely by fluorophore photobleaching (10,16).…”

Section: Discussionmentioning

confidence: 99%

“…Biochemical analysis has not identified the location or mode of preQ 1 binding, and the backbone flexibility of both RBS and anti-RBS sequences did not modulate appreciably as a function of preQ 1 concentration (23), unlike class I and II preQ 1 riboswitches that show clear preQ 1 -dependent RBS sequestration (16,18,32,33).…”

mentioning

confidence: 99%

“…The preQ 1 -I, preQ 1 -II, S-adenosyl-L-methionine-II (SAM-II), and fluoride riboswitches are representative of this organizational strategy, and their analysis has contributed to a renaissance in our understanding of regulatory pseudoknot structure and dynamics (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). By contrast, pseudoknotted aptamers that do not integrate their expression platforms are less common, and this added complexity can encumber efforts to elucidate how ligand-induced conformational changes regulate gene expression.…”

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confidence: 99%

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Structural analysis of a class III preQ ₁ riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Liberman

Suddala

Aytenfisu

et al. 2015

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

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PreQ 1 -III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ 1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HL out -type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ 1 -III riboswitch aptamer forms a HL out -type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ 1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ 1 binds tightly (K D,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3′ RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ 1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (k dock ∼0.6 s −1 ) and undocking (k undock ∼1.1 s −1 ). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs. preQ 1 riboswitch | gene regulation | crystal structure | single-molecule FRET | molecular dynamics R iboswitches are structured RNA motifs that sense the cellular levels of small molecules to provide feedback regulation of genes (1). Although present in all domains of life, they are prominent in bacteria where they typically reside in the 5′-leader sequences of mRNA (2). Broad interest in riboswitches originates from the discovery that they can be targeted by antimicrobials (3-5), and the observation that they use complex scaffolds to achieve gene regulation without the need for protein partners. In the latter respect, riboswitches typically exhibit bipartite sequence organization comprising a conserved aptamer linked to a downstream expression platform (2). Aptamer binding to a cognate effector can induce conformational changes that alter the accessibility of expression platform sequences, such as those required for transcriptional read-through, or hybridization to the 16S rRNA as a preface to translation (2, 6).Numerous riboswitches fold as pseudoknots that conform to the H-type or closely related HL out -type topology, which have emerged as the most efficient RNA scaffolds to integrate aptamer and expression platform sequences (7). The preQ 1 -I, preQ 1 -II, S-adenosyl-L-methionine-II (SAM-II), and fluoride riboswitches are representative of this organizational strategy, and their analysis has contributed to a renaissance in our understanding of regulatory pseudoknot structure and dynamics (8-18). By contrast, pseudoknotted aptamers that do not integr...

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Section: Resultssupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structural analysis of a class III preQ ₁ riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Liberman

Suddala

Aytenfisu

et al. 2015

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

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“…These two classes employ distinct ligand binding modes (Figure 1) [30,31]. The class-I riboswitch recognizes preQ 1 with cytosine (C15) through classical Watson–Crick base pairing and additionally through bidentate interaction of the ligand’s N3 and C2-NH 2 with the trans Watson–Crick face of adenosine (A29) (Figure 1A,C).…”

Section: Resultsmentioning

confidence: 99%

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ₁riboswitches inE.coli

Neuner¹,

Frener²,

Lusser

et al. 2018

RNA Biology

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For this study, we utilized class-I and class-II preQ1-sensing riboswitches as model systems to decipher the structure-activity relationship of rationally designed ligand derivatives in vitro and in vivo. We found that synthetic preQ1 ligands with amino-modified side chains that protrude from the ligand-encapsulating binding pocket, and thereby potentially interact with the phosphate backbone in their protonated form, retain or even increase binding affinity for the riboswitches in vitro. They, however, led to significantly lower riboswitch activities in a reporter system in vivo in E. coli. Importantly, when we substituted the amino- by azido-modified side chains, the cellular activities of the ligands were restored for the class-I conditional gene expression system and even improved for the class-II counterpart. Kinetic analysis of ligand binding in vitro revealed enhanced on-rates for amino-modified derivatives while they were attenuated for azido-modified variants. This shows that neither high affinities nor fast on-rates are necessarily translated into efficient cellular activities. Taken together, our comprehensive study interconnects in vitro kinetics and in vitro thermodynamics of RNA-ligand binding with the ligands’ in vivo performance and thereby encourages azido- rather than amino-functionalized design for enhanced cellular activity.

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Tails of Three Knotty Switches: HowPreQ₁Riboswitch Structures Control Protein Translation

Belashov

Dutta

Salim

et al. 2015

Encyclopedia of Life Sciences

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Riboswitches are structured RNA molecules that regulate genes by sensing cellular levels of metabolites such as preQ 1 (7‐aminomethyl‐7‐deazaguanine). This economical platform is found predominantly in the 5′‐leader sequences of bacterial mRNAs, attracting attention as a potential antibiotic target. Members of the preQ 1 riboswitch family provide exemplary insights into translational regulation because their high‐resolution structures have been determined, providing a framework to interpret complementary single‐molecule, computational and biochemical analyses. Although class I and II preQ 1 riboswitches possess divergent pseudoknot architectures, both appear to function by burying the Shine‐Dalgarno sequence (SDS) within a core aptamer domain upon preQ 1 binding. By contrast, the class III preQ 1 riboswitch binds its ligand in an aptamer distant from the SDS. PreQ 1 binding increases the population of riboswitches that transiently sequester the SDS, representing an alternative regulatory paradigm. Progress on preQ 1 riboswitches is described, along with expectations for interfacing a riboswitch with the ribosome for translation initiation. Key Concepts Riboswitches are structured non‐protein‐coding RNAs that bind small molecules, tRNA or ions to control gene expression without the need for proteins. Riboswitches bind protein enzyme co‐factors suggesting that they are remnants of an ancient RNA world. Three classes of riboswitches bind the metabolite prequeuosine 1 (preQ 1 ), a pyrrolopyrimidine made exclusively in bacteria as a precursor to the hypermodified base queuosine, which is used widely in the biosphere to confer translational fidelity. PreQ 1 riboswitches adopt pseudoknot folds in which ligand binding stabilises coaxial helical stacking. High‐resolution structures of class I, II and III preQ 1 riboswitches reveal their overall three‐dimensional folds, along with chemical details that explain their high‐affinity ligand binding. Structural observations suggest that riboswitches co‐opt traditional RNA tertiary interactions, such as major‐groove base‐triples and A‐minor motifs, for specialised biological functions. Class I and II preQ 1 riboswitches integrate Shine‐Dalgarno sequences (SDS) into their aptamer cores, producing gene off states in response to preQ 1 binding. Class III preQ 1 riboswitches fold with spatially disparate aptamer and expression platform sequences that do not commingle in response to ligand binding. Single‐molecule fluorescence resonance energy transfer (smFRET) provides insight into ligand‐dependent conformational changes of preQ 1 riboswitches that control gene expression. Modelling suggests that the class II preQ 1 riboswitch must unfold only its anti‐SDS⋅SDS helix to interact with the 16S ribosomal RNA.

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Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold

Cited by 79 publications

References 35 publications

Structural analysis of a class III preQ ₁ riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Structural analysis of a class III preQ ₁ riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ₁riboswitches inE.coli

Tails of Three Knotty Switches: HowPreQ₁Riboswitch Structures Control Protein Translation

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Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold

Cited by 79 publications

References 35 publications

Structural analysis of a class III preQ 1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Structural analysis of a class III preQ 1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1riboswitches inE.coli

Tails of Three Knotty Switches: HowPreQ1Riboswitch Structures Control Protein Translation

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Structural analysis of a class III preQ ₁ riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Structural analysis of a class III preQ ₁ riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ₁riboswitches inE.coli

Tails of Three Knotty Switches: HowPreQ₁Riboswitch Structures Control Protein Translation