Ligand Response of Guanidine-IV riboswitch at Single-molecule Level

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.94706.2

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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 (red) 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) smFRET histograms and transition density plots of Cy3Cy5-riboG-term 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) 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). (D) The percentages of the folded conformation of Cy3Cy5-riboG-term change with Mg²⁺ at 0 (black) and 1.0 mM Gua⁺ (green). (E) The percentages of the folded conformation of Cy3Cy5-riboG-term change with Gua⁺ at 0 (blue) and 10.0 mM Mg²⁺ (orange).

smFRET studies of the isolated full-length riboG at different concentrations of Mg²⁺ and Gua⁺.
(A) The secondary structures of Cy3Cy5-ful-length riboG at the unfolded (left) and the folded state (right). The full-length riboG contains the aptamer domain (black), terminator (red) and the extended sequence (blue). Cy3 and Cy5 are shown by green and red sparkles, respectively. (B) smFRET histograms and transition density plots for Cy3Cy5-full-length riboG 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) The percentages of the folded conformation of Cy3Cy5-full-length riboG change with Gua⁺ at 0 (green) and 10.0 mM Mg²⁺ (orange). (D–E) The normalized percentages of the folded conformation of Cy3Cy5-riboG-apt (black), Cy3Cy5-riboG-term (red) and Cy3Cy5-full-length riboG (blue) at 0–50 mM Mg²⁺ (D) and 0–100.0 mM Gua⁺ in the presence of 10.0 mM Mg²⁺ (E).

Relative free energy (ΔΔG) and kinetics analysis of isolated riboG-apt, riboG-term and full-length 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 full-length riboG (blue) in the presence of 0 and 1.0 mM Gua⁺ at 2.0 mM Mg²⁺. (C–D) Representative time traces of riboG-apt 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 T7 RNA polymerase. (B) Schematic diagram for smFRET experiment for ECs. The ECs were 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).

Ligand-mediated conformational switching of riboG during transcription.
(A) The schematic diagram of synthesizing ECs with nascent RNAs by in vitro transcriptional pause using PLOR reactions. The orange ellipse represents T7 RNA polymerase. (B) The secondary structures of full-length riboG. The halting 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, PAGE images of the crude products collected at the last step of individual PLOR strategy in the absence and presence of 1.0 mM Gua⁺. FL and T are represented for the read-through and terminated RNA. The detail strategies were depicted in Figure 7–figure supplement 1B. D, The transcriptional read-through fractions were plotted with the last halting sites in 9 step-, 11 step-, 12 step-, 13 step-, 14 step-, 15 step-, 16 step- and 17 step-PLOR. The orange and blue curves are in the absence and presence of 1.0 mM Gua⁺. Taking into consideration that the 17 step-PLOR reaction exhibited a pause within the terminator region, resulting in a significant amount of terminated product at step 16, crude products from steps 16 and 17 were collected for (C) and (D) of the 17 step-PLOR reaction (Lanes 15 and 16 in C).

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, the nucleotides in termination sequence are shown as red dots in EC-89 and EC-105, and DNA templates from EC-87 to EC-105 are not displayed in the model. The T7 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 green 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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