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

Abstract

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...

Molecular mechanism for preQ1-II riboswitch function revealed by molecular dynamics

“…Upon ligand binding, this riboswitch buries the SD sequence in an H-type (HL out ) pseudoknot, and this conformation is hypothesized to attenuate translation by preventing binding by the ribosome. High-resolution structures have been determined for the preQ 1 -I, preQ 1 -II, and preQ 1 -III translational riboswitches in the ligand-bound state (Spitale et al 2009;Jenkins et al 2011;Liberman et al 2013Liberman et al , 2015Kang et al 2014). The structures of the apo state, however, have only been determined for the preQ 1 -I riboswitch (Jenkins et al 2011), leaving open the question of how the preQ 1 -II riboswitch changes its conformation in the absence of the preQ 1 ligand.…”

“…More specifically, MD has elucidated chemical features that stabilize ligand binding, the interactions that stabilize conformational endpoints, and features that facilitate conformational switching. Studies have been conducted on the SAM-I riboswitch (Huang et al 2009;Stoddard et al 2010;Doshi et al 2012;Hayes et al 2012;Huang et al 2013;Xue et al 2015), the SAM-II riboswitch (Kelley and Hamelberg 2010;Doshi et al 2012), the guanine riboswitch (Villa et al 2009;Sund et al 2014), the adenine riboswitch (Sharma et al 2009;Priyakumar and MacKerell 2010;Nozinovic et al 2014;Sund et al 2014), the GlmS riboswitch (Banáš et al 2010;Xin and Hamelberg 2010), and the preQ 1 -I and preQ 1 -III riboswitches (Petrone et al 2011;Banáš et al 2012;Eichhorn et al 2012;Gong et al 2014;Liberman et al 2015). Overall, these studies show the feasibility of using MD to understand riboswitch function and provide an opportunity to look for common themes in ligand binding and conformational dynamics.…”

Regulatory effects of cotranscriptional RNA structure formation and transitions

“…Changes in many RNA structures regulate gene expression at the transcriptional level, resulting in either transcription termination or activation. Structural changes of some riboswitches upon ligand binding can regulate RNA transcription . In the adenine riboswitch cotranscriptional folding mechanism, the aptamer domain is formed prior to the expression domain by RNA polymerase, and the nascent aptamer domain is able to response to cellular cues and potentially bind to a cognate ligand before the transcription and folding of the expression domain .…”

“…Structural changes of some riboswitches upon ligand binding can regulate RNA transcription. 16,42 In the adenine riboswitch cotranscriptional folding mechanism, the aptamer domain is formed prior to the expression domain by RNA polymerase, and the nascent aptamer domain is able to response to cellular cues and potentially bind to a cognate ligand before the transcription and folding of the expression domain. 16,31 The binding of an adenine riboswitch ligand to an aptamer domain enables formation of an intrinsic terminator RNA hairpin stem structure, which inhibits RNA polymerase extension.…”

Nanomechanics and co-transcriptional folding of Spinach and Mango

“…The light-up aptamers integrate the distinct structural characteristics of RNA GQs with their ligand binding ability. Although folding of RNA aptamers found in riboswitches has been studied extensively [31][32][33][34] , biophysical characterizations of light-up aptamers regarding their folding properties are sparse 35,36 . For example, in order to function efficiently in cells, an RNA aptamer must fold correctly and stably and resist inadvertent unfolding induced by mechanical forces exerted by the fluctuating environments or by RNA binding or RNA-translocating proteins.…”