Differences between cotranscriptional and free riboswitch folding.

Area of Science:

• Molecular Biology
• Computational Biology
• Biophysics

Background:

• Riboswitches are noncoding RNA elements that regulate gene expression by sensing small molecules.
• Ligand binding induces conformational changes in riboswitches, influencing downstream gene activity.
• Simultaneous RNA polymerase (RNAP) elongation, folding, and ligand binding occur on a seconds' timescale.

Purpose of the Study:

• To investigate how transcript elongation velocity impacts the folding pathways of S-adenosylmethionine (SAM)-I and adenine riboswitches.
• To compare folding characteristics under free-folding versus transcription-rate limited conditions.
• To determine if physiological transcription rates limit in vivo riboswitch folding.

Main Methods:

• Employed two complementary coarse-grained in silico techniques: molecular dynamics (MD) simulations and kinetic Monte Carlo (kMC) simulations.
• MD simulations provided 3D, atomically resolved folding models with homogenous energetics.
• kMC simulations offered energetically detailed, longer-timescale analysis on the secondary structure level, including competing conformations and ligand binding.

Main Results:

• Observed and quantified distinct RNA structure formation pathways under different extrusion scenarios, with strong agreement between MD and kMC methods.
• Demonstrated that free-folding riboswitches exhibit different folding characteristics compared to transcription-rate limited folding.
• Identified a critical transcription rate distinguishing these folding regimes, which was found to be higher than physiologically relevant rates.

Conclusions:

• The folding of SAM-I and adenine riboswitches is influenced by transcript elongation velocity.
• In vivo folding of these riboswitches is likely limited by the rate of transcription.
• Computational modeling provides robust insights into the complex dynamics of riboswitch folding during transcription.