Probing and perturbing riboswitch folding using a fluorescent base analogue

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Biology

Background:

• Riboswitches are regulatory mRNA elements controlling gene expression.
• The Class I preQ1 riboswitch forms a pseudoknot structure upon ligand binding.
• Adenine-rich sequences in the L3 domain are crucial for preQ1 riboswitch function.

Purpose of the Study:

• To investigate the role of adenine residues in the L3 domain of the preQ1 riboswitch.
• To assess the impact of 2-aminopurine (2-AP) substitution on ligand binding and riboswitch structure.
• To understand how alterations in L3 affect preQ1 recognition.

Main Methods:

• Site-specific insertion of 2-aminopurine (2-AP) into the L3 domain of the preQ1 riboswitch.
• Fluorescence spectroscopy to monitor 2-AP fluorescence.
• Circular dichroism (CD) spectroscopy to analyze global conformational changes.
• Ligand titration experiments to determine binding affinities.

Main Results:

• 2-AP substitution reduced preQ1 binding affinity at all tested positions.
• Global conformational changes occurred similarly in modified and unmodified riboswitches at high ligand concentrations.
• Proximity to the ligand-binding pocket influenced the impact of 2-AP on binding affinity.
• Distal 2-AP sites affected L3 domain stability in the absence of ligand.
• Multiple 2-AP insertions did not cause further significant disruption.

Conclusions:

• Adenine residues within the L3 domain are essential for preQ1 ligand recognition.
• 2-AP substitution provides insights into the complex roles of these adenine residues.
• The study highlights the intricate interplay between sequence, structure, and function in riboswitch-mediated gene regulation.