Full-Length NAD⁺-I Riboswitches Bind a Single Cofactor but Cannot Discriminate against Adenosine Triphosphate

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Biology

Background:

• Bacterial riboswitches regulate gene expression by binding small molecules.
• Two classes of riboswitches sense nicotinamide adenine dinucleotide (NAD+), a crucial redox cofactor.
• The NAD+-I riboswitch has two homologous domains, but its full-length structure and function remain unclear.

Purpose of the Study:

• To investigate the cofactor binding affinity and specificity of individual domains and the full-length bacterial NAD+-I riboswitch.
• To clarify the mechanism of NAD+ sensing by this riboswitch class.

Main Methods:

• Isothermal titration calorimetry (ITC) was used to quantify binding affinities.
• Gel-filtration chromatography was employed to assess domain self-association.
• Ligand binding stoichiometry was determined for various dinucleotides.

Main Results:

• Domain I of the NAD+-I riboswitch binds NAD+ with a K_D of 24.6 μM, but exhibits nonproductive self-association.
• Domain II showed no detectable binding to NAD+.
• The full-length riboswitch binds a single NAD+ with a K_D of 31.5 μM and unexpectedly binds ATP with higher affinity (K_D = 11.0 μM).
• The binding affinity order for the full-length riboswitch is ADP = ATP > NAD+ = AP2-ribavirin > NADH.

Conclusions:

• The full-length NAD+-I riboswitch binds NAD+ at physiologically relevant concentrations.
• The observed binding of ATP and other dinucleotides suggests lower specificity than previously assumed.
• Further research is needed to understand the gene regulatory implications of this dual-binding capability.