Magnesium controls aptamer-expression platform switching in the SAM-I riboswitch

Abstract

Investigations of most riboswitches remain confined to the ligand-binding aptamer domain. However, during the riboswitch mediated transcription regulation process, the aptamer domain and the expression platform compete for a shared strand. If the expression platform dominates, an anti-terminator helix is formed, and the transcription process is active (ON state). When the aptamer dominates, transcription is terminated (OFF state). Here, we use an expression platform switching experimental assay and structure-based electrostatic simulations to investigate this ON-OFF transition of the full length SAM-I riboswitch and its magnesium concentration dependence. Interestingly, we find the ratio of the OFF population to the ON population to vary non-monotonically as magnesium concentration increases. Upon addition of magnesium, the aptamer domain pre-organizes, populating the OFF state, but only up to an intermediate magnesium concentration level. Higher magnesium concentration preferentially stabilizes the anti-terminator helix, populating the ON state, relatively destabilizing the OFF state. Magnesium mediated aptamer-expression platform domain closure explains this relative destabilization of the OFF state at higher magnesium concentration. Our study reveals the functional potential of magnesium in controlling transcription of its downstream genes and underscores the importance of a narrow concentration regime near the physiological magnesium concentration ranges, striking a balance between the OFF and ON states in bacterial gene regulation.

Figure 1.

Secondary and tertiary structure of full-length SAM-I riboswitch (with sequence) in SAM-bound transcription OFF state and SAM-free transcription ON state. (A) Sequence-aligned secondary structure and (B) tertiary structure of the transcription OFF state of SAM-I riboswitch in the presence of metabolite, SAM (yellow pentagon) surrounded by explicit magnesium ions (purple). Different secondary structural segments are defined sequence-wise. Note the partially overlapped aptamer and EP (EP) domains. (C) Sequence-aligned secondary structure and (D) tertiary structures of the transcription ON state surrounded by explicit magnesium (purple) ions. Four characteristic segments, important for switching, are designated with distinct colors: Red: switching strand; green: terminator helix in the EP domain; black: flexible aptamer; gray: more stable aptamer. In the transcription OFF state the flexible aptamer owns the red switching strand. In the transcription ON state green terminator sequesters the red switching strand.

Figure 2.

Experimental and computational investigations of [Mg²⁺] dependence of aptamer domain and the same for full-length SAM-I riboswitch (aptamer+EP). (A) SHAPE probing location at the J_1/4 region that connects four helices in the aptamer domain. (B) Experimental (black triangle) and simulated (red triangle) SHAPE reactivity measurements as a function of Mg²⁺ concentration. The data are best fitted to a Hill equation which quantifies the collapse transition midpoint, [Mg²⁺]_1/2. Experimental and simulation measurements yield close [Mg²⁺]_1/2 value (∼0.3–0.4 mM). (C) Schematic of experiment: EP is added to aptamer RNA for various magnesium concentrations. An RNA oligomer mimics the operation of native EP sequence, which is capable of sequestering switching strand sequence from the aptamer when the aptamer is less stable. (D) An electrophoretic mobility shift assay was used to calculate the ratio of the peak area from the aptamer in the OFF-state to the peak area for the aptamer–anti-terminator complex in the ON state as a function of [Mg²⁺]. (E) Theoretically computed ratio of the same was calculated from the free energy landscape of the transition between transcription ON and transcription OFF state. The population profile as a function of [Mg²⁺] shows non-monotonic dependence with a maximum transcription OFF state population ∼4.0 mM Mg²⁺.

Figure 3.

Mg²⁺ dependence of transcription ON-OFF conformational transition of SAM-I riboswitch. (A) Free energy landscape of transcription ON–OFF transition in the absence and in the presence of metabolite, SAM (0.4 mM). The reaction coordinate is the difference in the number of native contacts that distinguish the transcription ON state (number of contacts between the aptamer and the switching strand (N_transON)) from the transcription OFF state (number of contacts between the terminator and the switching strand (N_transOFF)). Note, SAM-induced stabilization of the transcription OFF state relative to the transcription ON state in the free energy landscape. (B) Mg²⁺ concentration dependence of transcription ON-OFF transition of SAM-I riboswitch in the absence of SAM. Average free energy profiles at varied [Mg²⁺] show different stability difference between the transcription ON state and the transcription OFF state. The stability difference between the transcription ON and the transcription OFF states (ΔG_{transOFF-transON}) quantified as a function of [Mg²⁺] has a non-monotonic [Mg²⁺] dependence, as shown in the inset. The aptamer reaches its maximum stability at around 4.0 mM Mg²⁺ range. Beyond 4.0 mM it starts to destabilize. Along with the stable transcription ON and OFF states (A and E, respectively), the transition involves different intermediates (B, C1, C2, D), which also have different Mg²⁺ sensitivity.

Figure 4.

Transcription ON–OFF transition routes of full-length SAM-I riboswitch. Two-dimensional free energy landscape as a function of transcription ON reaction coordinate and OFF reaction coordinate, exploring two distinct pathways at lower and higher Mg²⁺ concentrations. (A) At lower concentration ([Mg²⁺] = 0.1 mM), transition predominantly follows an orthogonal path via an entropically stable intermediate (C1). (B) At higher concentration ([Mg²⁺] = 10.0 mM), it preferentially follows a coordinated path via an enthalpically stabilized intermediate (C2). (C) Representative structures. The representative structure corresponding to each minimum of the energy landscape and serving as landmark on the orthogonal and coordinated pathways.

Figure 5.

Thermodynamic and structural origin of aptamer destabilization. (A) Non-monotonic [Mg²⁺] dependence of thermodynamic quantities reflects that initial OFF state stabilization up to 4.0 mM is due to comparatively stiff enthalpic stabilization, while destabilization of the OFF state beyond 4.0 mM is due to relatively stiff entropy decrease. This entropy-led OFF-state destabilization restricts the pre-organization of the aptamer within 2.0–4.0 mM concentration window. Aptamer destabilization is caused by Mg²⁺ mediated domain closure. (B) The Mg²⁺ dependent domain closure is quantified with the distance between the aptamer and the EP (terminator (T) helix). (C) The representative OFF-state structure extracted from our simulated trajectory equilibrated at 0.1 mM. It shows the aptamer and expression-platform domain separation. (D) The representative OFF-state structure from 10.0 mM Mg²⁺ simulation. It shows the aptamer and the expression-platform domain (terminator (T) helix) closure within the structure where the aptamer, the anti-terminator and the terminator helices partially co-exist and restrain the dynamics attaining the maximum collapse. Magnesium mediated backbone phosphate-phosphate contact map at Mg²⁺ concentration (E) 0.1 mM and (F) 10.0 mM. The newly formed magnesium mediated contacts at 10.0 mM are highlighted by a rectangular box that distinguishes P3-T contacts.

Figure 6.

Comparative Mg²⁺ dependence of individual aptamer and anti-terminator domains demonstrates that the stable anti-terminator helix formation is triggered beyond 2.0 mM. (A) Average free energy profiles of folding transition of the flexible aptamer domain as a function of transcription OFF reaction coordinates at different [Mg²⁺] show Mg²⁺ induced collapse transition occurs within 0.1–2.0 mM [Mg²⁺] range. (B) The representative structure corresponding to each minimum of the energy landscape is designated as follows: (i) ligand-free partially closed (PC), (ii) ligand-free partially open (PO) and an (iii) open (O) state minimum. (C) Average free energy profiles of folding transition of the anti-terminator helix as a function of transcription ON reaction coordinates at different [Mg²⁺] show Mg²⁺ induced collapse transition occurs within 2.0–4.0 mM [Mg²⁺] range. It reflects that compared to the aptamer domain, the anti-terminator helix requires higher critical Mg²⁺ concentration to promote its collapse transition. (D) The representative structure corresponding to each minimum of the free energy landscape of the anti-terminator helix folding is designated as follows: (i) closed (C), (ii) partially open (PO) and an (iii) open (O) state minimum.