Single-molecule Ligand Response of Guanidine-IV riboswitch

Lingzhi Gao
Dian Chen
Yu Liu author has email address

State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
Shanghai Artificial Intelligence Laboratory, Shanghai 200232, China

https://doi.org/10.7554/eLife.94706.1

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Figures and data

Characteristics of the C. botulinum guanidine-IV riboswitch.
(A, B) The secondary structures of the transcriptional termination and read-through states of the guanidine-IV riboswitch. The full-length of the guanidine-IV riboswitch contains the aptamer domain (black), terminator (orange) and the extended sequence (blue). The green nucleotides are involved in forming a KL in the anti-terminator state. (C) A transcriptional model of the guanidine-IV riboswitch. The DNA is shown in blue ribbons. The colors in the RNA are encoded as in (A) and (B). And the addition of Gua⁺ facilitates transcription read-through. (D) The percentages of the transcription read-through plotted with 0.5 μM–20.0 mM Gua⁺ at 6.0 mM Mg²⁺ in the three independent transcription termination reactions.

smFRET studies of post-transcriptional Cy3Cy5-riboG-apt at different concentrations of Mg²⁺ and Gua⁺.
(A) The secondary structures of Cy3Cy5-riboG-apt at the unfolded (left) and the folded state (right). Cy3 and Cy5 are shown by green and red sparkles, respectively. (B) smFRET histograms and transition density plots for Cy3Cy5-riboG-apt at 0 mM Mg²⁺, at 2.0 mM Mg²⁺, at 1.0 mM Gua⁺, and at 2.0 mM Mg²⁺ and 1.0 mM Gua⁺. (C) HMM analysis of smFRET time trace of Cy3Cy5-riboG-apt at 2.0 mM Mg²⁺, the time resolution is 0.1s. (D) Schematic diagram for three conformations of Cy3Cy5-labeled RNA in smFRET experiments. The Cy3Cy5-labeled RNA is hybridized with a biotinylated DNA and immobilized on the slides. (E) The percentages of the folded conformation (E_FRET ∼ 0.8) of Cy3Cy5-riboG-apt at 0–50.0 mM Mg²⁺ (cyan columns) and 0.5–100.0 mM Gua⁺ (red columns). (F) The percentages of the folded conformation of Cy3Cy5-riboG-apt change with Gua⁺ at 0, 0.5, 1.0 and 2.0 mM Mg²⁺. (G) FRET histograms of Cy3Cy5-riboG-apt at 100.0 mM KCl (black curve), NaCl (green curve), urea (red curve) and guanidine (blue curve). The experiments were repeated for three times.

smFRET studies of post-transcriptional riboG-term at different concentrations of Mg²⁺ and Gua⁺.
(A) The secondary structures of Cy3Cy5-riboG-term at the unfolded (left) and the folded state (right). (B–E) smFRET histograms and transition density plots of Cy3Cy5-riboG-term at 0 mM Mg²⁺ (B), 2.0 mM Mg²⁺ (C), 1.0 mM Gua⁺ (D), and 2.0 mM Mg²⁺ and 1.0 mM Gua⁺ (E). (F) The percentages of the folded conformation (E_FRET ∼ 0.8) of Cy3Cy5-riboG-term at 0–50.0 mM Mg²⁺ (cyan columns) and 1–100.0 mM Gua⁺ (red columns). (G) The percentages of the folded conformation of Cy3Cy5-riboG-term change with Gua⁺ at 0 (blue) and 10.0 mM Mg²⁺ (orange). (H) The percentages of the folded conformation of Cy3Cy5-riboG-term change with Mg²⁺ at 0 (black) and 1.0 mM Gua⁺ (green).

smFRET studies of the isolated riboG at different concentrations of Mg²⁺ and Gua⁺.
(A) The secondary structures of Cy3Cy5-riboG at the unfolded (left) and the folded state (right). (B–E) smFRET histograms and transition density plots for Cy3Cy5-riboG at 0 mM Mg²⁺ (b), 2.0 mM Mg²⁺ (C), 1.0 mM Gua⁺ (D), and 2.0 mM Mg²⁺ and 1.0 mM Gua⁺ (E). (F) The percentages of the folded conformation of Cy3Cy5-riboG change with Gua⁺ at 0 (green) and 10.0 mM Mg²⁺ (orange). (G–H) The normalized percentages of the folded conformation of Cy3Cy5-riboG-apt (black), Cy3Cy5-riboG-term (red) and Cy3Cy5-riboG (blue) at 0–50 mM Mg²⁺ (G) and 0–100.0 mM Gua⁺ in the presence of 10.0 mM Mg²⁺ (H).

Relative free energy (ΔΔG) and kinetics analysis of isolated riboG-apt, riboG-term and riboG.
(A) The relative free energy of the pre-folded and folded states of riboG-apt in the presence of 0 and 1.0 mM Gua⁺ at 2.0 mM Mg²⁺. The free energy of the unfolded state was referred as control. (B) The relative free energy of the pre-folded and folded states of riboG-term (orange) and riboG (blue) in the presence of 0 and 1.0 mM Gua⁺ at 2.0 mM Mg²⁺. (C–D) Representative time traces at 0 (C) and 1.0 mM Gua⁺ (D) in the presence of 2.0 mM Mg²⁺. Δτ is the dwell time. (E) The lifetime (τ) and rate constant (k) of unfolded (black), pre-folded (orange) and folded (blue) states of riboG-apt was determined by exponential decays of the dwell time distributions. (F) Schematic diagram of riboG-apt folding and transcriptional processes at 2.0 mM Mg²⁺ in the presence of 0 mM Gua⁺ (red) and 1.0 mM Gua⁺ (blue).

smFRET studies of nascent riboG in ECs without and with Gua⁺ at 0.5 mM Mg²⁺.
(A) The ECs containing an RNA at the unfolded state (left) and the folded state (right). The pause sites of EC-87 to EC-105 are marked by black arrows, and the orange ellipse represents the RNA polymerase. (B) Schematic diagram for smFRET experiment for ECs. The ECs are immobilized on the slides by their biotinylated DNA templates. (C–I) smFRET histograms for the EC-87 to EC-105 at 0 mM (orange) and 10.0 mM Gua⁺ (blue) at 0.5 mM Mg²⁺. (J) The normalized increase of the folded conformation (E_FRET ∼ 0.8) of ECs upon the addition of 1.0 mM Gua⁺ at 0.5 mM Mg²⁺ (grey), 10.0 mM Gua⁺ at 0.5 mM Mg²⁺ (green) and 1.0 mM Gua⁺ at 2.0 mM Mg²⁺ (red). (K) The denaturing PAGE image of EC-88 elongation assay after overnight at room temperature.

In vitro transcriptional performance of riboG.
(A) The schematic diagram of synthesizing ECs with nascent RNAs by in vitro transcriptional pause using PLOR reactions. The orange ellipse represents RNA polymerase. (B) The secondary structures of riboG. The pause sites of EC-69, EC-77, EC-78, EC-83, EC-88, EC-91, EC-94 and EC-105 are marked by red dots. (C) The denaturing PAGE image of EC-69 to EC-105. The slow migration from EC-69 to EC-105 matches the growing lengths of RNAs. (D) The increase of transcriptional read-through upon the addition of 1 mM Gua+ with the last pause site at 69, 77, 78, 83, 88, 91, 94 and 105, respectively before completing transcription.

Folding model of RiboG in the absence and presence of guanidine.
The folding pathways of anti-terminator conformation in the presence of Gua⁺ and terminator conformation in the absence of Gua⁺ are highlighted in blue and orange, respectively. The H-bonds in the KL are shown as dotted green lines. The nucleotides from EC-87 to EC-88 are shown as black dots, and the nucleotides in termination sequence are shown as red dots in EC-89 and EC-105. The RNAP is shown as orange ellipse. The DNA templates are shown as gray ribbons. Green arrows represent the direction of structural switching in native riboG upon the addition of the Gua⁺. The thicker the arrow, the higher the switching percentages. And the Gua⁺-sensitive transcription window of riboG from EC-87 to EC-88 is boxed by green lines.

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