PMC

Folding analysis based on Kinetic MC simulations. (A) Fraction of base pairs formed in each helix (normalized regional Q-value) as a function of the total number of formed base pairs for free folding of the adenine and SAM-I riboswitch secondary structures. (B) Folding of each helix over the chain growth rate for the SAM-I and adenine riboswitches. Folding events are characterized by the number of base pairs formed in the whole structure when a normalized regional Q-value of 0.5 is reached (mid Q-value, B).

Folding analysis of the extended sequence of the SAM-I riboswitch using Kinetic MC simulations. (A) Normalized regional Q-value as a function of time for free folding. (B) Normalized regional Q-value as a function of time for the chain growing at a transcription rate of ≈ 50 nt/s. (C) Probability (color coded) to find AT formed at the time when the complete chain is fully grown as a function of the transcription rate and . (D) Normalized regional Q-value as a function of time for the chain growing at a transcription rate of ≈ 50 nt/s. Here the multiloop is stabilized by to mimic the effect of the ligand.

Tertiary and secondary structures of the SAM-I and adenine riboswitches in ligand bound state. (A) Aptamer region of the SAM-I riboswitch (PDB ID 2GIS): the colored strands indicate elements of secondary structure, helix P1 in red, P2 in green, P3 in blue and P4 in pink. The ligand is shown in orange. (B) Aptamer region of the add adenine riboswitch (PDB ID 1Y26). The same colors are used as in (A) for helices P1 to P3 and ligand. (C) The SAM-I riboswitch consists of two pairs of coaxially stacked helices P1 to P4 connected by a four way helical junction in its ligand bound state. Helix P1 forms in the presence of the ligand and acts as an antiantiterminator allowing the terminator (long-stem loop with downstream sequence of uridines) to fold. In this case, transcription is terminated. (D) The add adenine riboswitch exhibits three helices P1 to P3 in its ligand bound state two of which are coaxially stacked. Helix P1 forms in the presence of the ligand and prevents a translational repressor (initiation codon paired in long-stem loop) from forming.

Schematics of the setups for SBM and kinetic MC simulations. (A) Schematics of the setup for an SBM simulation. The tube with a funnel-like exit region composed of a helix of SBM atoms surrounds the stretched RNA. The tube/atoms are positioned on a helix with a diameter d₁ of 20 Å and a length L₁ of 1100 Å to contain the whole stretched RNA strand. The exit funnel has a length L₂ of 40 Å and an outer diameter d₂ of 30 Å. These are the spatial constraints that prevent folding before the riboswitch has left the RNAP. Forces acting between the rear end of the tube (red ring) and every tenth nucleotide (red circles) extrude the RNA strand out of the tube with a constant rate. Whenever a nucleotide leaves the tube, it is released of its acting force and therefore free to fold mimicking the natural sequential transcription process. (B) Schematics of the kinetic MC method. The kinetic MC method grows the RNA chain with a constant rate allowing more and more base pairs to form or open in the available sequence.

Folding analysis based on SBM simulations. (A) Folding pathways of the SAM-I and adenine riboswitch for free folding. The plots for each riboswitch are based on 180 folding trajectories, starting from a stretched RNA strand. The mean values and standard deviations are plotted. We can derive the folding order by condensing a substructure’s curve in a single value representing the mid Q-value. (B) Folding events of substructural elements over the extrusion rate. A folding event is characterized by the number of formed helical base pairs at a normalized regional Q-value of 0.5, the mid Q-value. The regional Q-value data are gathered from 80 trajectories for each extrusion rate. The nonlocal helix P1 ties up both ends of the sequence and, therefore, folds last in both riboswitches. Both riboswitches fold in order of appearance in the limit of slow extrusion rates. We investigate a wide range of extrusion rates to cover the natural range of transcription rates. An extrusion rate of 0.0025 corresponds to an estimated transcription rate of ≈ 20 nt/s.