The conformational landscape of transcription intermediates involved in the regulation of the ZMP-sensing riboswitch from Thermosinus carboxydivorans

Abstract

Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity.

INTRODUCTION

Riboswitches are cis-acting regulatory RNA elements, which sense the concentration variation of metabolites of low molecular weight. They are found in the 5′-untranslated region of mRNA (1), primarily within prokaryotes (2). In general, riboswitches consist of a phylogenetically conserved aptamer domain that undergoes conformational change upon binding a specific ligand, which induces a further allosteric conformational change in a downstream expression platform (3). More than 20 different classes of riboswitches have been identified and have been found to sense metabolites including thiamin pyrophosphate (4,5), adenosylcobalamin (6,7), S-adenosylmethionine (8,9) and flavin mononucleotide (10). Typically, ligands sensed by riboswitches are cell metabolites but exceptions including riboswitches sensing fluoride (11) or Mg²⁺ (12) are known. Various riboswitches bind nucleobases such as adenine (13) and guanine (14) or the nucleoside deoxyguanosine (15). Often, riboswitches are found upstream of genes coding for proteins involved in the metabolism or catabolism of the ligand resulting in a feedback loop (16).

Transcriptional riboswitches modulate gene expression during transcription by controlling the formation of rho-independent terminator structures (17). Structural rearrangement of the aptamer domain upon binding leads to formation or destabilization of an antiterminator structure, competing with the terminator structure.

Despite the abundance of crystal structures of metabolite-bound aptamer domains, high-resolution structural information about the full range of transcriptional intermediates and their potentially heterogeneous conformations in the context of co-transcriptional folding are sparse. Switching efficiency between functional ON- and OFF-states in transcriptional riboswitches strongly depends on the time window during which the switch adopts a binding-competent form. In the Bacillus subtilis FMN riboswitch, transcriptional pausing is an important mechanism to achieve kinetic control (18). Time-resolved NMR experiments in our laboratory on two different purine-sensing riboswitches, the guanine-sensing riboswitch from B. subtilis and the 2′-deoxyguanosine-sensing riboswitch from Mesoplasma florum showed that these riboswitches exhibit kinetic control of regulation and the required structural transition to ensure both, functional ON- and OFF-states, are matched to the time window available during transcript elongation (18,19).

The function of second messengers in cells is long-known (20), but their role in riboswitch-based regulation was not identified until the late 2000s when Breaker et al. showed that gene expression is regulated by riboswitches that bind cyclic di-GMP (21). Several other conserved second messenger-sensing motifs were identified in the following years, including the pfl motif which binds the cellular alarmone ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR)) (22).

The ZMP-sensing riboswitch was identified in 2015 and its aptamer fold was named pfl motif, as it is frequently associated with a gene coding for pyruvate-formate-lyase (23). As a precursor of inosine, ZMP is involved in the purine and folate biocycle where it is converted into FAICAR by addition of one carbon, provided by 10f-THF. In case of increasing folate stress, ZMP acts as an alarmone, upregulating the expression of associated proteins (24). Regulation is achieved by riboswitches containing the pfl motif, which specifically recognize ZMP and ZTP. High ZMP concentrations induce ZMP-binding to the riboswitch, which leads to antiterminator formation and subsequent activation of gene expression. The ZMP-switch is thus considered a transcriptional ON-Switch.

The pfl motif consists of three helical stems with P2 located in the loop of P1 (Figure 1A). Junction J1/2, located between those helices, forms a pseudoknot with the loop of P3, which features a complementary sequence. Upon ligand binding, the pseudoknot is further stabilized by stacking interactions with ZMP. From the crystal structures it is known that the aromatic ring of ZMP stacks right below the pseudoknot, where it interacts with a U residue of the loop capping P3 (25). Without ZMP, nucleotides within the P3 stem base-pair to downstream residues as transcription proceeds, forming a rho-independent terminator hairpin. The stabilized pseudoknot thus acts as antiterminator, preventing formation of the terminator hairpin (24).

Figure 1.

Overview of the secondary structures of the ZMP-sensing riboswitch from T. carboxydivorans. (A) Secondary structure of the ZMP-sensing riboswitch in pseudoknot form with the construct Zsw⁷⁷ highlighted in blue. Investigated transcriptional intermediates are annotated accordingly. (B) Secondary structures of constructs Zsw⁴⁸ and Zsw¹⁵ used for investigation of pseudoknot formation. (C) Secondary structure of Zsw¹⁰¹, terminator fold of the ZMP-sensing riboswitch. Stems P1, P2, P3, the Junction J1/2 and the terminator hairpin T are annotated.

The linker between P1 and P3 is not conserved in length and cannot be resolved in crystal strutures due to structural heterogeneity. Studies by Ferré-d’Amaré et al. showed that removal of the linker does not abrogate ZMP binding, though a 10-fold lowered affinity is observed (26).

In a recent study, Lucks et al. applied SHAPE-seq (27) to the ZMP-sensing riboswitch from Clostridium beijerinckii and described its folding landscape in presence and absence of ZMP (28). They found that riboswitch function is ensured by a labile balance between the conserved short P3 stem, terminator hairpin and pseudoknot stability, which can be strongly perturbed by alteration of a single base-pair in one of those key-players.

In the present study, we characterize the conformations of transcriptional intermediates of the ZMP-sensing riboswitch from T. carboxydivorans under equilibrium conditions. These intermediates include the 48 nt containing construct Zsw⁴⁸ encompassing stems P1 and P2, the 77 nt construct Zsw⁷⁷ including stems P1, P2, and P3, ten transcriptional intermediates (Zsw⁸¹-Zsw⁹¹, Figure 1A) capable to switch between antiterminator and terminator conformation as well as the full-length riboswitch Zsw¹⁰¹. We use NMR spectroscopy as outlined in Fuertig et al. (29) to determine the conformations of transcriptional intermediates under varying conditions, employing rapid sample preparation methodology developed by Helmling et al. (30) ensuring homogeneity in transcript length. Further, an in-trans RNA construct consisting of isolated P1–P2 (Zsw⁴⁸) and P3 (Zsw¹⁵) was investigated to monitor pseudoknot formation in trans (Figure 1B), utilizing differential isotopic labeling for the two RNA constructs for isotope-filtered NMR experiments.

We provide a detailed conformational characterization of the states that are relevant for regulation and that are sufficiently stable to be populated during transcription in order to extend simple two state models describing function of transcriptional ON-switches.

MATERIALS AND METHODS

Transcription templates

Transcription templates were generated by PCR from pUC57 vector that contains the full-length native sequence of the ZMP-sensing riboswitch from T. carboxydivorans or from previously PCR-amplified DNA. Reverse primers used to obtain templates for Zsw⁴⁸, Zsw⁷⁷ and Zsw^81–91 are summarized in Table S1 (see Supplementary Material). Primers with high annealing temperatures exceeding 60 °C were chosen for Zsw¹⁰¹ as they led to significantly better PCR results. Primers for Zsw^81–91 contained 2′-methoxy modifications to ensure 3′-end homogeneous transcription products (30). PCR was performed according to the standard protocol by New England Biolabs^® (0.5 mM of each primer, 200 mM dNTPs) using home-made Phusion polymerase. As DNA template, we used either 0.1 μl of 1 μM plasmid solution containing the full-length riboswitch sequence or 0.1 μl of PCR-amplified template. Sample integrity was confirmed by native PAGE or agarose gel electrophoresis.

RNA preparation

All RNAs except Zsw¹⁵ were prepared by in vitro transcription with T7 RNA polymerase (RNAP). PCR-reactions were directly used for transcription without purification. Transcription reactions contained transcription buffer (100 mM Tris/glutamate pH 8.1), 2 mM spermidine, 20 mM dithiothreitol (DTT), 20% (v/v) DMSO, 5 mM of each NTP, 12.5 mM Mg(OAc)₂, 0.2 u/ml yeast inorganic pyrophosphatase (New England Biolabs^®) and 144 nM home-made T7 RNAP. Unlabeled NTPs were purchased from Carl Roth GmbH + Co. KG (Germany). ¹³C,¹⁵N labeled NTPs were purchased from Silantes GmbH (Germany).

The transcripts Zsw^81–91 were purified according to the protocol developed by Helmling et al. (30). After transcription in 10 ml scale, the RNA was washed on 5000 MWCO centrifugal concentrators (Vivaspin 20® from Sartorius AG, Germany). After loading the RNA on the centrifugal concentrator, the first two washing steps were performed with transcription buffer, to remove excess phosphate. Afterwards, the RNA was concentrated to 1 ml and washed at least 10 times with NMR buffer (25 mM potassium phosphate buffer, pH 6.2, 50 mM KCl) in 5 ml steps. Final NMR samples were concentrated to a volume of 250 μl and concentrations ranging from 80 to 120 μM. The RNA was thermally refolded before sample preparation by heating at 60°C for 5 min, subsequent addition of desired amount of magnesium chloride, heating at 40°C for 5min and cooling on ice for 30 min.

The RNA constructs Zsw⁷⁷ and Zsw⁴⁸ were purified by denaturing PAGE. The transcription mixture was loaded on a 3000 MWCO centrifugal concentrator (Vivaspin 20® from Sartorius AG, Germany) and washed repeatedly with water until the elute did not show a significant signal at 180–220 nm in the UV-VIS spectrum (NanoDrop One, ThermoFischer Scientific). The solution was concentrated to 1 ml, mixed with 30 % glycerol and loaded onto a 15% polyacrylamide (PAA) gel (7 M urea). The RNA band was excised from the gel and eluted in 0.6 M NaOAc, pH 5.5. The RNA was precipitated once with cold EtOH and twice with 2 % (w/v) LiClO₄ in acetone. After resuspending in water, buffer exchange to NMR buffer (as described above) was performed on a centrifugal concentrator.

Zsw¹⁵ RNA was bought from Dharmacon GmbH. The RNA was deprotected according to the supplier protocol. Following HPLC purification, the HPLC buffer was removed on a 1000 MWCO centrifugal concentrator. After one EtOH and two LiClO₄ precipitations (see above), the RNA was resuspended in water and the buffer exchanged to NMR buffer. The NMR buffer contained 25 mM potassium phosphate buffer adjusted to a pH of 6.2 and 50 mM KCl. The RNA was thermally refolded before sample preparation.

NMR spectroscopy

NMR samples were prepared by adding 10 % D₂O and 7.5 nmol DSS as internal reference to RNA stock solutions in NMR buffer (see above). All spectra were recorded of 280 μl samples in Shigemi NMR tubes (Shigemi Inc.).

NMR experiments were conducted on Bruker AV600, AV700 and AV800 spectrometers, equipped with cryogenic probes. Data were processed with Bruker Topspin 3.5 (Bruker Biospin) and sparky 3.14 (31). Water suppression was achieved using WATERGATE (32) or jump-and-return echo (33) water suppression pulse schemes. ¹⁵N-editing in ¹H,¹H-NOESY was achieved by implementing X-filter schemes before and after t₁ chemical shift evolution (34). Analysis of dissociation constants and binding competent fraction were carried out by measuring intensities of the imino proton signal of U42 in ¹H-1D spectra, normalization of values and subsequent fitting while leaving the RNA concentration variable.

ITC measurements

RNA samples for ITC measurements were essentially prepared as for NMR spectroscopy. ITC measurements were performed on a Malvern^® MicroCal iTC200 instrument with RNA sample concentrations of 60 μM and ligand concentrations of 600 μM (for Zsw⁷⁷ and Zsw⁹¹) or 300 μM (for constructs Zsw^81–83). Raw data were exported and analyzed using NITPIC (35) and SEDPHAT (36). Fractions bound were obtained from leaving the n-value variable in the fitting procedure.

RESULTS

NMR resonance assignment

NMR signals of longer RNA constructs can often be dissected to arise from structural elements already present in shorter constructs following a divide-and-conquer-strategy (29). Since stems P1 and P2 are present in Zsw⁴⁸ and in Zsw⁷⁷ (see Figure 1A and b), comparable sets of imino signals are expected for these constructs and could in fact be observed in ¹H-1D as well as ¹H,¹H-2D NOESY experiments (Figure 2). By contrast, NMR signals at 10.5, 11.6 and 12.8 ppm, observable in Zsw¹⁵, were also observed in Zsw⁷⁷ but not in Zsw⁴⁸.

Figure 2.

Imino regions of ¹H,¹H-NOESY spectra of Zsw⁴⁸ (A) and Zsw⁷⁷ (B) annotated with assignment. Color-coding indicates resonances from either P1 (cyan) or P2 (orange). Data were measured at 600 MHz, 2048 × 640 points and 184 scans and 800 MHz, 2048 × 512 and 256 points for Zsw⁴⁸ and Zsw⁷⁷, respectively. Samples contained 800 μM (Zsw⁴⁸) or 400 μM (Zsw⁷⁷) RNA, 25 mM potassium phosphate buffer (pH 6.2), 50 mM KCl and 10 % D₂O. Larger representations of the spectra are shown in Supplementary Figure S1 (C) Imino to aromatic region of the ¹H,¹H-NOESY of Zsw⁴⁸ (see A) and ¹⁵N-SOFAST-HMQC (¹⁵N-SFHMQC) of Zsw⁴⁸, showing ¹⁵N chemical shift differences for G and U residues. ¹⁵N-SOFAST-HMQC was measured at 700 MHz, room temperature, 2048 × 128 points and 16 scans on a sample containing 400 μM ¹³C, ¹⁵N-labeled RNA in 25 mM potassium phosphate buffer (pH 6.2), 50 mM KCl and 10 % D₂O. Subtle differences in chemical shift are a result of slight temperature differences between spectrometers.

For ¹H proton chemical shift assignment, ¹H,¹H-NOESY experiments were conducted mapping out the interaction of imino protons with adjacent protons less than 5 Å apart from each other and led to the sequential assignment of G and U residues in P1 and P2 stems. Distinction between signals arising from G and U residues, respectively, was achieved by analysis of the cross-signals to the opposing base, which are two C amino protons for G-C base-pairs, but only one aromatic adenine H2 proton for A-U base pairs; and confirmed by ¹H,¹⁵N-correlation spectra on an isotopically labeled sample of Zsw⁴⁸ (Figure 2C). Imino protons of the terminal helical base-pairs in P1 and P2 could not be observed due to solvent exchange. Since the P2 helix is not involved in ligand interaction or pseudoknot formation, we omitted further characterization of this stem. For helix P3 in Zsw⁷⁷ and Zsw¹⁵ (Figure 1B), no sequential imino proton NOEs were observed, most likely due to the rapid solvent exchange in this very short stem. Subsequently, the assignment of Zsw⁴⁸ was transferred to Zsw⁷⁷ (Figure 2A and B). In summary, we established the conformations for both Zsw⁷⁷ and Zsw⁴⁸. Further imino proton signals were observed at 12.1 and 12.7 ppm for Zsw⁴⁸ and an additional signal at 12.3 ppm for Zsw⁷⁷. These signals likely arise from residues located in the non-helical regions between P1 and P2, since they show no NOESY cross peaks to other imino proton signals.

Investigation of pseudoknot interaction via in-trans construct

To investigate the formation of the pseudoknot between J1/2 and P3, which is the essential interaction stabilizing the antiterminator conformation, we designed two RNA constructs Zsw⁴⁸ and Zsw¹⁵. Zsw⁴⁸ consisted of the P1 and P2 stem including the junction J1/2, and Zsw¹⁵ consisted of the P3 helix with the corresponding loop. To assess whether these two RNA strands reconstruct the aptamer domain in-trans, we added one equivalent of Zsw¹⁵ to Zsw⁴⁸ and monitored chemical shifts of imino proton signals in the presence of 10 mM Mg²⁺ and 2 eq. ZMP (Figure 3A). Imino proton spectra of the in-trans construct Zsw⁴⁸+Zsw¹⁵ showed no difference to spectra of Zsw⁷⁷, confirming that the pseudoknot formation does not require the linker J3. However, spectra also showed that ZMP affinity is higher for the in-cis construct Zsw⁷⁷ than for the in-trans construct Zsw⁴⁸+Zsw¹⁵. The linewidth of reporter signal U42 was larger for Zsw⁴⁸+Zsw¹⁵, indicating conformational heterogeneity involving a residual unbound conformation, while U42 in Zsw⁷⁷ showed a significant chemical shift perturbation (CSP) of 0.06 ppm. Strikingly, however, Zsw⁴⁸+Zsw¹⁵ showed clear ligand binding (Figure 3A).

Figure 3.

(A) ¹H-1D NMR spectra of the in-trans construct Zsw⁴⁸+Zsw¹⁵ and Zsw⁷⁷ before and after addition of 2 eq. ZMP in presence of 10 mM Mg²⁺. (B) ¹⁵N-edited and conventional ¹H-1D NMR spectra of ¹⁵N-Zsw⁴⁸+¹⁴N-Zsw¹⁵ and conventional ¹H-1D of Zsw¹⁵ in the presence of 10 mM Mg²⁺. Peaks that arise only in the conventional spectrum marked with arrows. Samples contained 100 μM RNA. Stacked and enlarged representations of those spectra are shown in Supplementary Figure S2. (C) Schematic representation of the labelling scheme of ¹⁵N-Zsw⁴⁸+¹⁴N-Zsw¹⁵. Colors of base-pairs according to arrow colors in (B). (D) 12 % native PAGE of Zsw⁴⁸ in the presence of 1eq Zsw¹⁵, 10 mM Mg²⁺ and 1 eq. ZMP. Pockets were loaded with 200 nmol Zsw⁴⁸ and additions as indicated. The gel is displayed enlarged in Supplementary Figure S3 (E) Top: ¹⁵N-SOFAST-HMQC spectrum (37) of Zsw⁴⁸ before (cyan) and after (black) addition of Zsw¹⁵ in the presence of 10 mM Mg²⁺. Spectra were recorded at 800 MHz at room temperature with 2048 × 128 points and 8 scans. Bottom: ¹⁵N-edited ¹H,¹H-NOESY of Zsw⁴⁸+Zsw¹⁵ with the cross peaks that arise from addition of Zsw¹⁵ marked blue. Spectra were recorded at 800 MHz with 2048 × 432 points and 256 scans.

We prepared ¹³C,¹⁵N-isotope labeled Zsw⁴⁸ and added unlabeled Zsw¹⁵ to further characterize the pseudoknot interaction. We recorded ¹⁵N-edited ¹H-1D spectra as well as conventional ¹H-1D spectra of all constructs (Figure 3B upper and middle spectra, respectively). In ¹⁵N-edited spectra, peaks of only those ¹H can be observed that are covalently bound to ¹⁵N, which includes only signals of residues in Zsw⁴⁸. In the ¹H-1D spectrum, several signals were observed which could not be attributed to Zsw⁴⁸ since they did appear only in conventional ¹H-1D-, but not in ¹⁵N-edited spectra. Of these, signals at 11.7 ppm and 12.7 ppm (Figure 3B, black arrows) were assigned to the stem imino protons of Zsw¹⁵, as they closely resemble the signals observed in pure Zsw¹⁵ (Figure 3B lower spectrum). Signals at 12 ppm and 12.8 ppm belonged to Watson-Crick type base-pairs between Zsw⁴⁸ and Zsw¹⁵ indicative of pseudoknot formation (Figure 3B, orange arrows). Following up on this observation, we compared ¹H,¹⁵N-correlation spectra before and after addition of Zsw¹⁵ and observed an additional imino resonance at 12 ppm obscured by another ¹H resonance, which had not been detected in 1D experiments and only appears upon addition of Zsw¹⁵ and therefor pseudoknot formation. A cross peak from this resonance to 12.8 ppm was observed in the ¹⁵N-edited ¹H,¹H-NOESY of ¹⁵N-Zsw⁴⁸+¹⁴N-Zsw¹⁵ (Figure 3E) proving the formation of two consecutive Watson-crick type G-C base-pairs of the pseudoknot.

These NMR results were supported by native PAGE. By native PAGE (Figure 3D), we detected a slower migration speed for Zsw⁴⁸+Zsw¹⁵ in comparison to pure Zsw⁴⁸ even without addition of Mg²⁺. Adding Mg²⁺ slightly reduced migration speed, while addition of ligand slightly enhanced migration speed. We attribute the slower migration speed with Mg²⁺ to alteration of ion strength and the slight enhancement upon ligand interaction to a slight compaction of the structure upon pseudoknot stabilization.

Probing ZMP-binding to transcriptional intermediates of increasing length

We investigated binding of ZMP to potentially critical transcriptional intermediates of the pfl riboswitch by NMR spectroscopy. In the crystal structure of the riboswitch (25), a Mg²⁺-ion is detected in close proximity to the binding pocket and ITC studies by Patel et al. suggested strong dependence of ZMP affinity on Mg²⁺ concentration (25). Thus, we assumed structural changes to take place upon addition not only of ZMP, but also of Mg²⁺, which were detectable as CSP on imino proton resonances. For two samples of Zsw⁷⁷, we added either 3 mM or 10 mM of MgCl₂ ([Mg²⁺]:[RNA] = 15/50:1), respectively, before adding two equivalents ([RNA]:[ZMP] = 1:2) of ligand. In both cases, CSPs to the downfield region were observed for all proton signals after addition of Mg²⁺. However, these shifts were accompanied by signal broadening at 10 mM Mg²⁺. After adding two equivalents of ligand, signal line widths decreased and the signals shifted non-uniformly for the 10 mM Mg²⁺ sample. In contrast, the 3 mM Mg²⁺ sample showed no spectral changes upon ZMP addition (Figure 4A). This confirms the observations previously reported (26). The Mg²⁺-induced increase in signal line widths was reversed by the addition of ZMP. The imino proton resonance of U42 represents a potent probe for ligand binding, as it is well separated from the other signals in the spectrum and close enough to the ligand binding pocket to show significant CSPs upon interaction. In the pure RNA sample, it was observed as a strong signal at 13.77 ppm (Figure 4B black spectra). However, upon addition of Mg²⁺, an additional signal appeared at 13.9 ppm with the two populations in an estimated 1:1 ratio (Figure 4B orange spectrum). Addition of ligand than led to loss of the signal at 13.77 ppm and increase of the signal at 13.9 ppm accompanied by a small downfield shift (Figure 4B cyan spectra). These effects might be attributed to the Mg²⁺-dependent pre-formation of a conformationally flexible binding-competent conformational state, which is subsequently stabilized by ligand interaction. When Mg²⁺ was added to Zsw⁴⁸ no second conformation or line broadening was observed (Supplementary Figure S4). For Zsw¹⁰¹, no response to ligand or magnesium could be detected (Supplementary Figure S5).

Figure 4.

(A) ¹H-1D NMR spectra of Zsw⁷⁷ before and after addition of Mg²⁺ and subsequent addition of 2 eq. ZMP at room temperature. Samples contained 100 μM RNA. (B) NOESY and ¹H-1D data showing an overlay of the Ligand and Mg²⁺ free (black), Mg²⁺ bound (orange, only 1D) and ligand bound (cyan) state of Zsw⁷⁷ at 288K. (C) ¹H-1D NMR spectra of transcripts Zsw^81,82,83 under influence of 10 mM Mg²⁺ before and after addition of 2 eq. ZMP. U42, which is indicative for binding, is marked with an arrow. ZMP binding is visible for Zsw⁸¹ and Zsw⁸² while no binding is observed for Zsw⁸³. Samples contained 120 μM RNA. Zooms of the regions marked grey in top spectra are displayed in bottom spectra. (D) Denaturing PAGE of RNAs Zsw¹⁵, Zsw⁴⁹, Zsw⁷⁶ and Zsw^81–83. Gel contained 12 % PAA and 7 M urea. The full gel is shown in Supplementary Figure S8.

We investigated transcriptional intermediates between the fully binding-competent Zsw⁷⁷ and the binding-incompetent Zsw¹⁰¹ RNA for ligand binding. Ligand binding is linked to pseudoknot formation, which competes with terminator stem formation at increasing transcript length. From investigation of the secondary structure we could deduce that the shortest potential construct, to form a closing base-pair of a terminator stem-loop is Zsw⁸¹, and no additional stabilization for the terminator conformation can be expected beyond Zsw⁹¹. Accordingly, titration experiments were performed on constructs of lengths between 81 nt and 91 nt, at 10 mM Mg²⁺ and up to 2 eq. ZMP. The constructs were prepared by shifting the reverse primer 3′-end from 5′ to 3′ of the template, achieving different lengths of template DNA for in vitro transcription by PCR. In vitro transcriptions according to the protocol of Helmling et al. (30) allowed quick and homogeneous RNA preparation as shown by denaturing PAGE (Figure 4D and Supplementary Figure S6).

NMR titration (Figure 4C) of Zsw⁸¹ showed similar CSPs as for Zsw⁷⁷. In the ZMP-free form, under influence of Mg²⁺, a structural equilibrium was observed with U42 signals at 13.77 and 13.9 ppm as in spectra of Zsw⁷⁷. As opposed to the 1:1 ratio observed in Zsw⁷⁷, the binding-competent state was less populated in Zsw⁸¹, observed from the lower intensity of the U42 signal at 13.9 ppm. Lucks et al. showed that the addition of a nucleotide enabling the formation of a one base-pair terminator stem partially destabilizes the antiterminator conformation (28). This effect would explain the spectral alterations observed in the ZMP-free samples of Zsw⁷⁷ and Zsw⁸¹. However, ligand binding was clearly detectable by chemical shift change of U42. In contrast to the spectrum of ligand-bound Zsw⁷⁷, considerable line-broadening was observed even in the bound state, which suggests population of two or multiple states with interconversion kinetics in the intermediate time regime. For Zsw⁸², the effect of ligand addition was already minor compared to Zsw⁸¹, observed as ∼50% lower differences in peak intensities. Alongside, we observed increased spectral complexity and line-broadening. Zsw⁸³, able to form 3 base-pairs of the terminator hairpin, showed a further shift of the conformational ensemble towards the terminator conformation. Adding ZMP did not lead to any significant structural alteration anymore, which indicates that the terminator conformation was already stable enough to fully outcompete the ligand-binding competent antiterminator conformation. This was also observed for ZMP titration to constructs Zsw^84–90, which did not show any spectral changes upon ligand addition (Supplementary Figure S7).

We performed ITC measurements (Figure 5A) on the RNA constructs which showed ligand binding according to NMR titration experiments to determine the equilibrium binding constants. In most cases, riboswitch aptamers exhibit a 1:1 stoichiometry for ligand binding. However, conformational equilibria between binding competent and binding incompetent folds, as observed by NMR, decrease the effective concentration of ligand-binding competent RNA conformations. Accordingly, the data were fitted to accommodate to possible binding incompetent fractions by leaving the stoichiometric parameter variable. Thus, not only the dissociation constants, but also the relative binding competent fractions of the RNA could be obtained. Dissociation constants for Zsw⁷⁷ and Zsw⁸¹ were 0.57 and 0.54 μM respectively, which is in good agreement with 1 μM obtained by Ferré-d’Amaré et al. for the Fusobacterium ulcerans ZMP-sensing riboswitch (26). Dissociation constants obtained from NMR measurements (Figure 5B) were slightly higher with 2.78 μM for Zsw⁷⁷ and 2.58 μM for Zsw⁸¹. While the affinity remains constant throughout the two different RNA constructs, the amount of binding competent RNA largely differs. By ITC, the binding-competent fractions are 53 % (Zsw⁷⁷) and 13% (Zsw⁸¹), while in NMR measurements, a decrease from 70% to 43% was observed (Figure 5C). However, the degree of decreased binding differs largely between the two methods ITC and NMR-spectroscopy with a relative decrease of roughly 75% in ITC and only 40% in NMR measurements from Zsw⁷⁷ to Zsw⁸¹. A possible explanation could be the slow time-scale of equilibration between measurements. While ZMP was added with few minutes of waiting time between injections in ITC, NMR experiments take around a factor of 10 longer. For Zsw⁸², only a very minor binding competent population was observable both in ITC and NMR titrations, which prevented curve fitting and thus the determination of a dissociation constant or the exact population of states. However, we estimated from curve shapes of constructs longer than 81 nt that the amount of binding competent RNA must be below 10 % in ITC measurements, up to Zsw⁹¹, which showed no interaction at all.

Figure 5.

(A) ITC titration curves of ZMP to constructs Zsw⁷⁷, Zsw^81–83 and Zsw⁹¹. Dissociation constants determined by the fit are annotated as applicable. (B) NMR titration curves of ZMP to constructs Zsw⁷⁷, Zsw^81–83 and Zsw⁹¹. (C) Table summarizing the results of the titration experiments. Values for Zsw⁸² and Zsw⁸³ were estimated from the curves displayed. Errors, obtained from the fitting procedures are displayed with the respective curve.

DISCUSSION

In this study, we explored the folding landscape of the ZMP-sensing riboswitch from T. carboxydivorans by NMR spectroscopy and ligand binding affinities by ITC. From ¹H,¹H-NOESY experiments imino proton assignment of Zsw⁴⁸ could be achieved and transferred to the longer construct Zsw⁷⁷, enabling the observation of ligand binding on the well separated resonance of U42. We additionally could confirm pre-formation of the pseudoknot and binding competence of an in-trans construct in which the linker between P1 and P3 was deleted. We observed only two G-residues of the possible four G–C base-pairs in the pseudoknot, suggesting a lower stability at one edge of the pseudoknot helix. Taking the crystal structure (25) into account, it is highly likely that G16–C62 and G17–C61 are less stable than G14–C65 and G15–C64 base-pairs, since G14 sits directly adjacent to the highly structured binding-pocket, while G17–C61 constitutes the closing base-pair of the pseudoknot helix. As reported previously (25), binding was only achieved at a high Mg²⁺ concentration of 10 mM, where substantial line broadening of NMR signals was observed, while the initial linewidth was regained after ZMP addition. Upon addition of 10 mM Mg²⁺, we additionally observed a structural equilibrium between two states, in which one state showed reporter peak shifts similar to the ligand-bound and the other one similar to the free state. Upon ligand interaction the system collapsed into the ligand bound state with minimal chemical shift alteration compared to the Mg²⁺-bound state. We therefore propose an additional Mg²⁺-dependent pre-formation of the binding pocket, which goes beyond just pseudoknot-formation, since pseudoknot formation is already observed without Mg²⁺ addition. However, pseudoknot formation is required for this additional Mg²⁺-stabilized state, since Zsw⁴⁸ does not show any spectral alteration upon Mg²⁺ addition, in the absence of Zsw¹⁵. The Mg²⁺ binding step can thus be considered an intermediate step, taking place after pseudoknot formation and facilitating ligand interaction and therefore final stabilization of the binding pocket.

Longer constructs showed only slight alteration in the population of these states in the presence of Mg²⁺. However, the amount of RNA which refolded to the ligand-bound state upon ZMP addition plummeted between Zsw⁸¹ and Zsw⁸², visible from quantitative NMR and ITC data. This further indicated that the existence of a Mg²⁺-induced pre-bound state is decoupled from the pseudoknot-terminator equilibrium, but both must be present to allow ligand binding. Lucks et al. reported that already the addition of a single nucleotide, competing with P3 helix stability (i.e. Zsw⁸¹), leads to partial ZMP binding incompetence, which we also observe via ITC and NMR spectroscopy. However, for this construct, absolute values differed largely between ITC and much slower NMR measurements. We therefore suspect very slow folding kinetics on a timescale of minutes between a binding incompetent pre-terminator state back to a binding competent state if binding competent RNA is removed from the equilibrium by ligand interaction. This would render the pre-terminator state kinetically trapped in a biological context. Consequently, it is expected that RNA displaying formation of a single terminator base-pair could already be predominantly binding incompetent in vivo, where fast progress of transcription limits the refolding time available. In comparison, Zsw⁸² displays strongly reduced binding competence and an almost full population of the terminator state, leaving Zsw⁸¹ as the only construct with considerable population of both states. Therefore, in a biological context, the transition to the binding incompetent state is expected to be comparably sharp in contrast to other examples like the deoxyguanosine-sensing riboswitch, which displays a smoother transition (19). The high stability of short pre-terminator constructs leads to a very short window for ligand interaction during transcription of the riboswitch, which is only possible while RNA of lengths 74–76 to 81 nt is accessible to refolding and therefor ligand interaction. Linker length between P3 and terminator stem could therefore be an important feature that tunes riboswitch activity over genetic occurrences as in other prokaryotes, where this length strongly varies (25). It should be noted, that in terms of co-transcriptional folding, 14 nucleotides, starting from the active side are involved in the transcription elongation complex (TEC), with 9 nt hybridized to DNA and 5 nt located in the exit channel (38). In principle 2 to 3 more residues could be involved in RNA folding and removed from the DNA:RNA duplex without the TEC dissociating (39), leaving 7 to 8 residues in the exit channel with potential for interaction. While these residues are unable to interact in tertiary interactions with further upstream residues, as in the pseudoknot structure, RNA secondary structure formation inside the exit channel is observed in some SHAPE-seq (40) and smFRET (41) experiments for specific constructs. The assessment of the ability of the Zsw terminator structure to form in the exit channel of the T. carboxydivorans RNAP is beyond the scope of this project. Therefore, nucleotide numbers mentioned here, refer to residues accessible to folding in a manner comparable to in vitro folding, so most likely outside the RNAP exit channel. Consequently, during folding of the first pre-terminator base-pair at position 81, the RNAP active site will reside at position 91–93, the position of the terminator poly-U stretch. Therefore, transcriptional speed might be additionally modulated by transcriptional pausing, a well studied effect in transcriptional regulation and action of rho-independent terminators (18,42).

We propose a model (Figure 6) in which a pre-formed pseudoknot is further stabilized by ligand interaction, leading to anti-termination of the riboswitch, which is in agreement with the results Lucks et al. obtained using SHAPE-seq (28). The time-window of the binding competent conformation is, due to the very stable pre-terminator Zsw⁸², comparably short, featuring structural equilibria only at a transcript length of 81 nt. This implies that for biological function to be carried out correctly, the binding event must be considerably fast compared to transcript elongation (possibly modulated by trans acting factors such as Nus proteins), which could be an interesting premise for further kinetic experiments. With the terminator fold being thermodynamically more stable than the ligand-bound antiterminator fold, the stabilization of the pseudoknot established by ZMP addition can be considered temporary and therefore kinetically driven. This type of regulation is a common motif observed in riboswitches such as fluoride- (43), FMN- (44), deoxyguanosine- (19) or guanosine-sensing (45) riboswitches. Biological function of the riboswitch is therefore expected to be heavily tuned by terminator folding kinetics with and without the influence of ZMP.

Figure 6.

Schematic representation of the model for genetic control employed by the ZMP-sensing riboswitch from T. carboxydivorans. The point of genetic decision during transcription of nucleotides 67–81 is characterized by binding competence. At this stage, the pseudoknot is preformed. Binding of the cognate ligand ZMP leads to stabilization of the pseudoknot bearing antiterminator structure and subsequent gene transcription. No binding leads to formation of the terminator structure and rho-independent transcription termination.

To resolve these kinetics, real-time NMR measurements can be performed by rapid addition of interactors or by introduction and subsequent elimination of photolabile protecting groups. However, these studies require sufficient exploration of the underlying system in equilibrium state. We herein provided the NMR spectroscopic basis for such studies, showing important key players of the conformational landscape of the pfl riboswitch from T. carboxydivorans and their structural characteristics at atomic resolution.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

DFG priority programme [SPP 1879]; collaborative research center [SFB902]; B.M.R.Z. is supported by the state of Hesse. Funding for open access charge: institutional funding.

Conflict of interest statement. None declared.