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Self-Cleavage of the Glms Ribozyme Core Is Controlled by a Fragile Folding Element

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Self-Cleavage of the Glms Ribozyme Core Is Controlled by a Fragile Folding Element Self-cleavage of the glmS ribozyme core is controlled by a fragile folding element Andrew Savinova and Steven M. Blockb,c,1 aBiophysics Program, Stanford University, Stanford, CA 94305; bDepartment of Applied Physics, Stanford University, Stanford, CA 94305; and cDepartment of Biology, Stanford University, Stanford, CA 94305 Edited by Ronald R. Breaker, Yale University, New Haven, CT, and approved October 15, 2018 (received for review July 13, 2018) Riboswitches modulate gene expression in response to small- biophysical study. We investigated this core domain using optical molecule ligands. Switching is generally thought to occur via the force spectroscopy, a powerful tool for studying the folding and stabilization of a specific RNA structure conferred by binding the activity of individual, structured RNAs (3, 15–18). Here, we re- cognate ligand. However, it is unclear whether any such stabili- port the free energy landscape traversed by the core glmS ribo- zation occurs for riboswitches whose ligands also play functional zyme during folding—from a fully unfolded ssRNA to a functional roles, such as the glmS ribozyme riboswitch, which undergoes self- ribozyme—and its relationship to catalytic activity. Measurement cleavage using its regulatory ligand, glucosamine 6-phosphate, as of the folding and biochemical behaviors of single molecules in a catalytic cofactor. To address this question, it is necessary to real time, under various ligand conditions, revealed a close in- determine both the conformational ensemble and its ligand de- terplay of structural dynamics, cofactor binding, and catalysis. pendence. We used optical tweezers to measure folding dynamics Our studies were conducted on a consensus core ribozyme and cleavage rates for the core glmS ribozyme over a range of construct (14) (Fig. 1A). Molecules of in vitro-transcribed core forces and ligand conditions. We found that the folding of a spe- glmS ribozyme were attached by their 5′ and 3′ ends to poly- cific structural element, the P2.2 duplex, controls active-site forma- tion and catalysis. However, the folded state is only weakly stable, styrene microspheres via DNA handles, facilitating measurement regardless of cofactor concentration, supplying a clear exception of the molecular extension under external forces produced by a to the ligand-based stabilization model of riboswitch function. dual-beam optical tweezers, using the so-called dumbbell assay (Materials and Methods and Fig. 1B). The unfolding of ribozyme BIOPHYSICS AND optical traps | single-molecule biophysics | folding | energy landscape constructs was investigated by performing a series of force-ramp COMPUTATIONAL BIOLOGY experiments, in which molecular extension was monitored with iboswitches are ligand-responsive, cis-regulatory mRNA el- nanometer-level precision as the distance between the two op- Rements found in all three domains of life (1, 2). Riboswitches tical traps was increased linearly with time. Worm-like chain generally function via either a kinetic or a thermodynamic (WLC) model fits to the fully folded and fully unfolded states in competition between alternatively folded RNA structures: an the resulting force-extension curves (FECs) (Fig. 1C) returned Δ = aptamer, which recognizes and binds to the cognate ligand, and the change in contour length due to complete unfolding, Lc an expression platform, which affects downstream gene expres- 34.5 ± 1.1 nm (Materials and Methods). This distance is consis- sion in one of several ways, for example by modulating tran- tent with release of all 66 nucleotides from a fully folded state scription or translation (1). In the conventional model of with an initial molecular width of 4.4 ± 1.1 nm (Materials and riboswitch function, the binding energy of the ligand is converted Methods), which is similar to the 3.3-nm width of the core do- into additional RNA folding stability, which favors the aptamer- main based on crystal structures (5, 6). bound configuration, holding the riboswitch in the “on” state (3, 4). However, not all riboswitches form alternatively folded Significance structures. A counterexample is provided by the bacterial glmS riboswitch, which has been found to fold into nearly the same Riboswitches are structured mRNA elements that regulate structure regardless of whether ligand is bound or not (5, 6). The gene expression in response to metabolite ligands. The bacte- glmS riboswitch regulates the expression of glucosamine-6- rial glmS riboswitch regulates a protein with a key role in cell phosphate synthase (GlmS), an enzyme that catalyzes synthesis wall biosynthesis. This riboswitch follows an unusual mecha- of the essential bacterial cell wall precursor, glucosamine 6- nism, acting as a ligand-activated self-cleaving ribozyme. Un- phosphate (GlcN6P) (7). Understandably, this riboswitch has derstanding how this RNA folds may aid development of become a target for antibiotic development (8, 9). Unique antibiotics targeting it. We used optical tweezers to investigate among the known riboswitches, the glmS riboswitch functions as this ribozyme riboswitch at the single-molecule level. We a self-cleaving ribozyme activated by its ligand, GlcN6P, which found that folding and function are intertwined in this system, also serves as a catalytic cofactor for the cleavage reaction (10, with a dynamically unstable folding transition controlling ca- 11). Cleavage generates a 5′-hydroxyl end that targets the glmS talysis. Furthermore, binding of the ligand fails to stabilize the mRNA for subsequent degradation by RNase J1 (12), thereby weakly favored folded state. Hence, the glmS ribozyme does down-regulating GlmS protein expression. The nearly identical not follow the conventional model for riboswitch switching, in structures of the ribozyme with and without cofactor bound which ligand binding stabilizes a particular switch conformation. presumably reflect equivalent low-energy states. However, prior work has not addressed the stability of the fully folded state or Author contributions: A.S. and S.M.B. designed research; A.S. performed research; A.S. how ligand binding might affect the dynamic ensemble of con- analyzed data; and A.S. and S.M.B. wrote the paper. formational states formed by this riboswitch. The authors declare no conflict of interest. The full glmS ribozyme carries a core domain, comprising a This article is a PNAS Direct Submission. double pseudoknot that forms the active site (5, 6), which is Published under the PNAS license. sufficient for GlcN6P-dependent self-cleavage activity (10, 13) 1To whom correspondence should be addressed. Email: [email protected]. (Fig. 1A). The isolated core domain folds from just 66 nucleo- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tides (14)—and potentially fewer, due to the P1 hairpin being of 1073/pnas.1812122115/-/DCSupplemental. variable length (11)—making it an attractive model system for www.pnas.org/cgi/doi/10.1073/pnas.1812122115 PNAS Latest Articles | 1of6 Downloaded by guest on September 25, 2021 A C 25 20 P1 P1 15 10 23 10 Force (pN) P2.2 5 Fig. 1. Optical tweezers measurements of core glmS P2.1 P2.1 ribozyme folding using the dumbbell assay. (A, Left) P2.2 30 42 0 Thermoanaerobacter tengcongensis glmS ribozyme 1 800 850 900 950 1000 1050 crystal structure, Protein Data Bank (PDB) ID code P2 Extension (nm) -1 2Z75, showing the duplex elements P1, P2, P2.1, and F = 12.9 pN P2.2 (core domain, dark colors; noncore domain, pale (5’) 60 D cyan). (A, Right) Secondary structure of the 66-nt P2 core construct used in this study (same color ssRNA linkers Extension scheme). (B) Cartoon of the optical trapping dumb- bell assay geometry (not to scale), showing the two Duplexes with (3’) 5 nm traps and beads attached to handles hybridized on DNA handles 0 0.2 either side of a core ribozyme molecule. (C) Repre- 2 s B sentative records of FECs for core glmS ribozyme Laser Beam F = 8.6 pN DNA Handles (solid colors), showing WLC model fits (dashed gray lines) (Materials and Methods) to the completely 0.82 µm (2 kb) (1 kb) 0.6 µm folded (low-force) and fully unfolded (high-force) Bead Bead Extension Dig / Biotin / states. (D) Representative records at constant force Anti-Dig Avidin (gray, raw data; red, median-smoothed), with the Core glmS Ribozyme 5 nm corresponding extension histograms show to the 0 0.2 right, fit to sums of Gaussians (red, histogram data; 10 s Time P(x) blue, individual Gaussians; black, sum of Gaussians). Higher-resolution details of the folding dynamics were in- stabilization of P2.2 formation was observed, with F1/2(apo) = vestigated by subjecting individual ribozymes to constant forces 5.7 ± 0.3 pN and F1/2(Glc6P) = 4.2 ± 0.7 pN (Fig. 2A). If anything, while measuring their molecular extensions over time (Materials the data indicate a minor destabilization induced by cofactor bind- and Methods). Performing such near-equilibrium measurements ing, but the observed effect is within 2σ. We confirmed that the across a range of fixed loads, we observed several distinct, absence of any ligand-induced stabilization was not specific to the thermally induced “hopping” transitions between folding states in cofactor analog we used by comparing the thermodynamics of P2.2 characteristic force regimes (Fig. 1D). The associated extension folding in the presence and absence of saturating levels of the changes, Δx, were determined for the transitions of each single- natural ligand, GlcN6P (10 mM), based on records obtained prior to molecule record by fitting a sum of Gaussians to the overall ex- self-cleavage events; these returned a similar value of F1/2(GlcN6P) = tension histogram (Fig. 1D). Likewise, the probabilities of folding 5.2 ± 0.7 pN (Fig. 2A). We also determined the average P2.2 folded for each transition, Pfolded, were determined from the folded-state and unfolded state lifetimes as functions of force (Materials and occupancies and fit to a Boltzmann function to determine the Methods) and found that the cofactor did not stabilize P2.2 kinetically corresponding F1/2 value, that is, the force where Pfolded = 0.5 (e.g., either,withnoincreaseinthefolded-statelifetimeobservedwhen Fig.
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