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Tuning a Riboswitch Response Through Structural Extension of a Pseudoknot

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Tuning a Riboswitch Response Through Structural Extension of a Pseudoknot Tuning a riboswitch response through structural PNAS PLUS extension of a pseudoknot Marie F. Soulièrea,1, Roger B. Altmanb,1, Veronika Schwarza, Andrea Hallera, Scott C. Blanchardb,2, and Ronald Micuraa,2 aInstitute of Organic Chemistry and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria; and bDepartment of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10021 Edited by David A. Tirrell, California Institute of Technology, Pasadena, CA, and approved July 23, 2013 (received for review March 8, 2013) Structural and dynamic features of RNA folding landscapes rep- been extensively investigated (18–28), preQ1 class II (preQ1-II) resent critical aspects of RNA function in the cell and are par- riboswitches have been largely overlooked despite the fact that ticularly central to riboswitch-mediated control of gene expression. a different mode of ligand binding has been postulated (14). Here, using single-molecule fluorescence energy transfer imaging, The consensus sequence and the secondary structure model we explore the folding dynamics of the preQ1 class II riboswitch, an for the preQ1-II motif (COG4708 RNA) (Fig. 1A) comprise upstream mRNA element that regulates downstream encoded mod- ∼80–100 nt (14). The minimal Streptococcus pneumoniae R6 fi i cation enzymes of queuosine biosynthesis. For reasons that are aptamer domain sequence binds preQ1 with submicromolar af- not presently understood, the classical pseudoknot fold of this sys- finity and consists of an RNA segment forming two stem–loops, tem harbors an extra stem–loop structure within its 3′-terminal re- P2 and P4, and a pseudoknot P3 (Fig. 1B). In-line probing gion immediately upstream of the Shine–Dalgarno sequence that studies suggest that the putative SD box (AGGAGA; Fig. 1) is contributes to formation of the ligand-bound state. By imaging li- sequestered by pseudoknot formation, which results in trans- gand-dependent preQ1 riboswitch folding from multiple structural lational-dependent gene regulation of the downstream gene (14). – fl perspectives, we reveal that the extra stem loop strongly in uences Here, we investigated folding and ligand recognition of the pseudoknot dynamics in a manner that decreases its propensity to S. pneumoniae R6 preQ1-II riboswitch, using complementary spontaneously fold and increases its responsiveness to ligand bind- chemical, biochemical, and biophysical methods including se- ing. We conclude that the extra stem–loop sensitizes this RNA to lective 2′-hydroxyl acylation analyzed by primer extension CHEMISTRY broaden the dynamic range of the ON/OFF regulatory switch. (SHAPE), mutational analysis experiments, 2-aminopurine fluo- rescence, and single-molecule fluorescence resonance energy SHAPE | single-molecule FRET | site-specific labeling | metabolite sensing transfer (smFRET) imaging. In so doing, we explored the struc- RNAs | ligand recognition tural and functional impact of the additional stem–loop element in the context of its otherwise “classical” H-type pseudoknot fold (29– variety of small metabolites have been found to regulate 32) (Fig. 1C). Our results reveal that the unique 3′-stem–loop el- Agene expression in bacteria, fungi, and plants via direct ement in the preQ -II riboswitch contributes to the process of interactions with distinct mRNA folds (1–4). In this form of 1 SD sequestration, and thus the regulation of gene expression, by BIOCHEMISTRY regulation, the target mRNA typically undergoes a structural – modulating both its intrinsic dynamics and its responsiveness to change in response to metabolite binding (5 9). These mRNA ligand binding. elements have thus been termed “riboswitches” and generally include both a metabolite-sensitive aptamer subdomain and an Results and Discussion expression platform. For riboswitches that regulate the process Temperature-Dependent SHAPE Indicates preQ -II Pseudoknot of translation, the expression platform minimally consists of 1 – Preorganization. Previous bioinformatics and in-line probing a ribosomal recognition site [Shine Dalgarno (SD)]. In the sim- studies on the preQ -II riboswitch sequence from S. pneumonia plest form, the SD sequence overlaps with the metabolite-sensitive 1 aptamer domain at its downstream end. Representative examples Significance include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as The three-dimensional fold and inherent structural dynamics of prequeuosine class I (preQ1-I) and II (preQ1-II) riboswitches (13, 14). The secondary structures of these four short RNA families RNA are critical determinants of function. Although the hier- contain a pseudoknot fold that is central to their gene regulation archical folding of RNA secondary structure followed by ter- tiary structure formation is well established, insights into how capacity. Although the SAM-II and preQ1-I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) secondary structure units impact tertiary structure formation and dynamics remain elusive. Using single-molecule FRET im- and preQ1-II counterparts are more complex and include a struc- aging of the preQ1 class II bacterial riboswitch, we reveal that tural extension that contributes to the pseudoknot architecture – (14). Importantly, the impact and evolutionary significance of RNA employs stem loop insertions into classical pseudoknots as a tool to tune ligand response and hence the outcome of these “extra” stem–loop elements on the function of the SAH and natural gene-regulatory elements. preQ1-II riboswitches remain unclear. PreQ riboswitches interact with the bacterial metabolite 1 Author contributions: M.F.S., S.C.B., and R.M. designed research; M.F.S., R.B.A., and V.S. 7-aminomethyl-7-deazaguanine (preQ1), a precursor molecule in performed research; A.H. contributed new reagents/analytic tools; M.F.S., R.B.A., V.S., the biosynthetic pathway of queuosine, a modified base en- A.H., S.C.B., and R.M. analyzed data; and M.F.S., S.C.B., and R.M. wrote the paper. countered at the wobble position of some transfer RNAs (14). The authors declare no conflict of interest. The general biological significance of studying the preQ1-II This article is a PNAS Direct Submission. system stems from the fact that this gene-regulatory element is Freely available online through the PNAS open access option. found almost exclusively in the Streptococcaceae bacterial family. 1M.F.S. and R.B.A. contributed equally to this work. Moreover, the preQ1 metabolite is not generated in humans and 2To whom correspondence may be addressed. E-mail: [email protected] or has to be acquired from the environment (14). Correspondingly, [email protected]. the preQ1-II riboswitch represents a putative target for antibiotic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. intervention. Although preQ1 class I (preQ1-I) riboswitches have 1073/pnas.1304585110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1304585110 PNAS Early Edition | 1of9 Downloaded by guest on September 25, 2021 exhibited enhanced SHAPE reactivity in the presence of preQ1 ligand (see ref. 35 and Fig. S3), suggesting that its solvent ex- posure increases as a consequence of preQ1 binding. Changes of this kind likely stem from disruption of the U19–A50 base pair in the ligand-bound complex. This conclusion is corroborated by the observation that a 2-aminopurine (2-Ap) nucleoside at po- sition 50 exhibits an increase in fluorescence in the presence of 2+ Mg and preQ1, trends that are in line with its movement from a stacked, or intrahelical, position to one that is extrahelical (35). Variations in denaturation temperature were also ob- Fig. 1. PreQ1 class II riboswitch. (A) Chemical structure of 7-aminomethyl- 7-deazaguanosine (preQ1); consensus sequence and secondary structure model for the COG4708 RNA motif (adapted from reference 14). Nucleoside presence and identity as indicated. (B) S. pneumoniae R6 preQ1-II RNA aptamer investigated in this study. (C) Schematics of an H-type pseudoknot with generally used nomenclature for comparison. R6 have yielded the putative secondary structure depicted in Fig. 1B (14). The characteristic pseudoknot (P3) interaction is de- fined by the pairing of the 3′-terminal sequence with junction J2–3, where the stem–loop P4 element is inserted immediately up- stream of this interaction. To explore the structural and dynamic properties of this model, we used the chemical probing technique SHAPE (33) to probe the conformation of the preQ1-II ribos- witch as a function of temperature. To do so, a transcribed riboswitch aptamer domain (85 nt in length; Materials and Methods and Fig. 2) was subjected to reaction with benzoyl cy- anide (BzCN) at temperatures ranging from 25 to 70 °C in 5 °C increments. After reverse transcription, the discrete bands ob- served by gel electrophoresis were quantified using SAFA soft- ware (Fig. S1) (34). The relative 2′-OH reactivities were plotted as a function of temperature to evaluate the denaturation tem- perature of the RNA at the single-nucleotide level (Fig. 2 A and B, and Fig. S2). Here, band intensities represent the degree of 2′- hydroxyl acylation of the base identified by reverse transcription. This analysis was repeated for the preQ1-II RNA in the absence or presence of magnesium ions and the preQ1 ligand. The results from these experiments are presented on the proposed second- ary structure and color-coded according to their
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