Confirmation of a second natural preQ<sub>1</sub> aptamer class in Streptococcaceae bacteria

Meyer, Michelle M.; Roth, Adam; Chervin, Stephanie M.; Garcia, George A.; Breaker, Ronald R.

doi:10.1261/rna.937308

Cited by 103 publications

(163 citation statements)

References 47 publications

(56 reference statements)

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“…At present, the preQ 1 -III riboswitch has been identified in a handful of Ruminococcaceae with the preponderance of sequences originating from metagenomes (23). The proximity of the aptamer to a downstream RBS preceding the queT start codon suggests a role in translational regulation comparable to many class I, and all class II preQ 1 riboswitches (23,32,33). However, at ∼100 nucleotides in length, the preQ 1 -III riboswitch is substantially larger than other family members, which exhibit more diminutive sizes ranging from 33 to 58 nucleotides.…”

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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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“…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).…”

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confidence: 99%

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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“…Some riboswitches require divalent cations for ligand binding (9, 12-16), whereas in others divalent cations stabilize the bound state (17-21). Some ligands, including S-adenosyl methionine (22), S-adenosyl homocysteine (22, 23), cyclic di-GMP (24, 25), and prequeuosine (preQ 1 ) (26, 27), have more than one class of riboswitch, with each class adopting a different 3D fold to achieve recognition of the same ligand.Two classes of riboswitches that bind preQ 1 , a precursor to queuosine, have been identified (26,27). Queuosine is a hypermodified guanine found universally at the anticodon wobble position of aspartyl, asparaginyl, histidyl, and tyrosyl tRNA (28) and is required for faithful translation (29-31).…”

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confidence: 99%

“…Some riboswitches require divalent cations for ligand binding (9,(12)(13)(14)(15)(16), whereas in others divalent cations stabilize the bound state (17)(18)(19)(20)(21). Some ligands, including S-adenosyl methionine (22), S-adenosyl homocysteine (22,23), cyclic di-GMP (24,25), and prequeuosine (preQ 1 ) (26,27), have more than one class of riboswitch, with each class adopting a different 3D fold to achieve recognition of the same ligand.…”

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Structural determinants for ligand capture by a class II preQ ₁ riboswitch

Kang

Eichhorn²,

Feigon

2014

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

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Prequeuosine (preQ 1 ) riboswitches are RNA regulatory elements located in the 5′ UTR of genes involved in the biosynthesis and transport of preQ 1 , a precursor of the modified base queuosine universally found in four tRNAs. The preQ 1 class II (preQ 1 -II) riboswitch regulates preQ 1 biosynthesis at the translational level. We present the solution NMR structure and conformational dynamics of the 59 nucleotide Streptococcus pneumoniae preQ 1 -II riboswitch bound to preQ 1 . Unlike in the preQ 1 class I (preQ 1 -I) riboswitch, divalent cations are required for high-affinity binding. The solution structure is an unusual H-type pseudoknot featuring a P4 hairpin embedded in loop 3, which forms a three-way junction with the other two stems. 13 C relaxation and residual dipolar coupling experiments revealed interhelical flexibility of P4. We found that the P4 helix and flanking adenine residues play crucial and unexpected roles in controlling pseudoknot formation and, in turn, sequestering the Shine-Dalgarno sequence. Aided by divalent cations, P4 is poised to act as a "screw cap" on preQ 1 recognition to block ligand exit and stabilize the binding pocket.Comparison of preQ 1 -I and preQ 1 -II riboswitch structures reveals that whereas both form H-type pseudoknots and recognize preQ 1 using one A, C, or U nucleotide from each of three loops, these nucleotides interact with preQ 1 differently, with preQ 1 inserting into different grooves. Our studies show that the preQ 1 -II riboswitch uses an unusual mechanism to harness exquisite control over queuosine metabolism.R iboswitches are functional noncoding RNA elements often found at the 5′ end of genes that bind to effector molecules, usually metabolites, to attenuate gene expression (1-8). They generally contain two modular elements, an aptamer that binds directly to a cognate ligand, followed by an expression platform that carries out the regulatory function. Ligand binding induces a conformational rearrangement in the aptamer domain, which is transduced to the expression platform, turning gene expression on or off at the transcription or translation level.Structural studies of diverse riboswitches bound to their cognate ligands have revealed that they have evolved various strategies for high-affinity and specific binding between RNA receptors and ligands. In general, the aptamer domain enfolds the ligand, forming a highly complex 3D conformation using a host of tertiary interactions, including base triples, kink turns, ribose zippers, A-minor motifs, loop-loop interactions, and pseudoknots (9-11). Some riboswitches require divalent cations for ligand binding (9, 12-16), whereas in others divalent cations stabilize the bound state (17-21). Some ligands, including S-adenosyl methionine (22), S-adenosyl homocysteine (22, 23), cyclic di-GMP (24, 25), and prequeuosine (preQ 1 ) (26, 27), have more than one class of riboswitch, with each class adopting a different 3D fold to achieve recognition of the same ligand.Two classes of riboswitches that bind preQ 1 , a precursor t...

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“…The preQ 1 -I (class 1) aptamer is distributed widely and rather compact, comprising not more than 34 nucleotides [28]. The preQ 1 -II (class 2) riboswitch is about twice this size and has been found in the Firmicutes [29]. Both classes are prevalent among important pathogens, such as Streptococcaceae .…”

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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Confirmation of a second natural preQ₁ aptamer class in Streptococcaceae bacteria

Cited by 103 publications

References 47 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

Structural determinants for ligand capture by a class II preQ ₁ riboswitch

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

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Confirmation of a second natural preQ1 aptamer class in Streptococcaceae bacteria

Cited by 103 publications

References 47 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

Structural determinants for ligand capture by a class II preQ 1 riboswitch

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

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Confirmation of a second natural preQ₁ aptamer class in Streptococcaceae bacteria

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

Structural determinants for ligand capture by a class II preQ ₁ riboswitch

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