Unraveling the structural complexity in a single-stranded RNA tail: implications for efficient ligand binding in the prequeuosine riboswitch

Abstract

Single-stranded RNAs (ssRNAs) are ubiquitous RNA elements that serve diverse functional roles. Much of our understanding of ssRNA conformational behavior is limited to structures in which ssRNA directly engages in tertiary interactions or is recognized by proteins. Little is known about the structural and dynamic behavior of free ssRNAs at atomic resolution. Here, we report the collaborative application of nuclear magnetic resonance (NMR) and replica exchange molecular dynamics (REMD) simulations to characterize the 12 nt ssRNA tail derived from the prequeuosine riboswitch. NMR carbon spin relaxation data and residual dipolar coupling measurements reveal a flexible yet stacked core adopting an A-form-like conformation, with the level of order decreasing toward the terminal ends. An A-to-C mutation within the polyadenine tract alters the observed dynamics consistent with the introduction of a dynamic kink. Pre-ordering of the tail may increase the efficacy of ligand binding above that achieved by a random-coil ssRNA. The REMD simulations recapitulate important trends in the NMR data, but suggest more internal motions than inferred from the NMR analysis. Our study unmasks a previously unappreciated level of complexity in ssRNA, which we believe will also serve as an excellent model system for testing and developing computational force fields.

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

“…We previously reported the dynamic properties of an isolated 12-nt ssRNA, hereafter referred to as SS, derived from a B. subtilis prequeuosine riboswitch (Eichhorn et al 2012a). We designed a ssRNA-helix junction construct variant containing the same 12-nt ssRNA connected to a GC-rich 22-bp hairpin capped with the thermodynamically stable cUUCGg tetraloop (E-SS) (Fig.…”

“…Recently we showed using a combination of NMR spectroscopy and replica exchange molecular dynamics (REMD) simulations that the adenine-rich, 12-nucleotide (nt) ssRNA in the Bacillus subtilis prequeuosine riboswitch adopts, on average, an A-form-like conformation with an ordered 6-nt adenine core and gradually increasing flexibility toward the terminal ends (Eichhorn et al 2012a). Together with CD and UV/Vis melting experiments, these data indicated that the ssRNA is in equilibrium between an ordered A-form-like helical conformation and a highly disordered partially melted state.…”

“…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.…”

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

“…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.…”

Tuning a riboswitch response through structural extension of a pseudoknot

“…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).…”