Folding of a transcriptionally acting PreQ ₁ riboswitch

Abstract

7-Aminomethyl-7-deazaguanine (preQ 1 ) sensitive mRNA domains belong to the smallest riboswitches known to date. Although recent efforts have revealed the three-dimensional architecture of the ligand-aptamer complex less is known about the molecular details of the ligand-induced response mechanism that modulates gene expression. We present an in vitro investigation on the ligand-induced folding process of the preQ 1 responsive RNA element from Fusobacterium nucleatum using biophysical methods, including fluorescence and NMR spectroscopy of site-specifically labeled riboswitch variants. We provide evidence that the full-length riboswitch domain adopts two different coexisting stem-loop structures in the expression platform. Upon addition of preQ 1 , the equilibrium of the competing hairpins is significantly shifted. This system therefore, represents a finely tunable antiterminator/terminator interplay that impacts the in vivo cellular response mechanism. A model is presented how a riboswitch that provides no obvious overlap between aptamer and terminator stem-loop solves this communication problem by involving bistable sequence determinants.RNA | chemical synthesis | labeling | bistable structures | riboswitches N oncoding regions of mRNA that bind metabolites with high selectivity and specificity function as so-called riboswitches (1-3). They represent gene regulation systems that are widespread among bacteria and importantly, they do not rely on the assistance of proteins (4). Riboswitches consist of a metabolite-sensitive aptamer and an adjoining expression platform (5) Although impressive progress has been made in revealing three-dimensional structures of metabolite-bound aptamer complexes of various riboswitch classes (6), less is known about how binding of the metabolite to the aptamer is communicated into a structural change of the expression platform, which in turn signals "on" or "off" for gene expression. The simplified picture of bacterial transcription control is that upon metabolite binding either a mRNA terminator structure is formed, which causes the RNA polymerase to stop synthesis (off regulation) or an existing terminator is disrupted, which enables the polymerase to continue synthesis with the mRNA template (on regulation) (5, 7). In the case of translational control, accessibility versus sequestration of the Shine-Dalgarno sequence upon metabolite binding is the essence of the response mechanism for on versus off regulation, corresponding to hindrance or enabling of bacterial ribosome translation initiation (5, 7-9).The preQ 1 class I riboswitch is the smallest riboswitch known to date (10). Its aptamer comprises a stretch of only 34 nucleotides in the 5′ untranslated leader region of the respective messenger RNA and specifically recognizes 7-aminomethyl-7-deazaguanine (preQ 1 ), which is an intermediate in queuosine biosynthesis (11). The minimal sequence and structure consensus refer to a hairpin comprising a five base-pair stem (P1) and a loop of eleven to thirteen nucleotides (L1) together...

Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

“…The kinetics of the structural changes from fold  A to fold  B ( k A→B ), and from fold  B to fold  A ( k B→A ), were determined from the single‐exponential fit of the distributions of τ Stem and τ Loop , respectively. The revealed kinetics of conformational switching between fold  A and fold  B structures were in the same order as for the preQ 1 induced structural switching rate obtained from stopped‐flow measurements 48. The addition of 1 μ m preQ 1 resulted in an increase in k A→B , while k B→A was almost unaffected.…”

Tuning a riboswitch response through structural extension of a pseudoknot

“…These estimates, which are based on local structural rearrangements of the RNA that report indirectly on the ligand binding process, are in line with the parameters reported previously for a number of other riboswitch aptamer domains (for comparison, see table S1 in ref. 22). Strikingly, the estimated k on for the truncated, ΔP4/A 3 construct was ∼1.6 ± 0.1 × 10 6 M −1 ·s −1…”

“…Correspondingly, the preQ 1 -II riboswitch represents a putative target for antibiotic intervention. Although preQ 1 class I (preQ 1 -I) riboswitches have been extensively investigated (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), preQ 1 class II (preQ 1 -II) riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated (14).…”

Structural dynamics of a single-stranded RNA–helix junction using NMR

“…While this study demonstrates the role of RNA sequence on interhelical dynamics and flexibility, little is known about the dynamic properties of ssRNA-helix junctions at the atomic level, even though they are often important sites for conformational changes and adaptation, particularly for systems containing pseudoknots (Kim et al 2008;Cao and Chen 2009;Cash et al 2013). The dynamic properties of ssRNA-helix junctions are of particular interest in the transcription-regulating prequeuosine riboswitch aptamer because the ssRNA tail must fold back onto the helix to form a pseudoknot to allow efficient cotranscriptional binding to ligand (Kang et al 2009;Klein et al 2009;Spitale et al 2009;Rieder et al 2010;Suddala et al 2013). Our previous studies on the isolated 12-nt ssRNA indicated a degree of order within the ssRNA that may facilitate rapid docking into the minor groove of the adjacent helix in the presence of ligand (Eichhorn et al 2012a); however, these studies neglected the effect of the ssRNA-helix junction.…”