Background: Cellular iron uptake, utilization, and storage are tightly controlled
through the action of iron regulatory proteins (IRPs). IRPs achieve this control by binding to
IREs-mRNA in the 5'- or 3'-end of mRNAs that encode proteins involved in iron metabolism.
The interaction of iron regulatory proteins with mRNAs containing an iron responsive
element plays a central role in this regulation. The IRE RNA family of mRNA regulatory
structures combines absolutely conserved protein binding sites with phylogenetically conserved
base pairs that are specific to each IREs and influence RNA/protein stability. Our
previous result revealed the binding and kinetics of IRE RNA with IRP1. The aim of the present
study is to gain further insight into the differences in protein/RNA stability as a function
of pH and ionic strength.
Objective: To determine the extent to which the binding affinity and stability of protein/RNA
complex was affected by ionic strength and pH.
Methods: Fluorescence spectroscopy was used to characterize IRE RNA-IRP protein interaction.
Results: Scatchard analysis revealed that the IRP1 protein binds to a single IRE RNA molecule.
The binding affinity of two IRE RNA/IRP was significantly changed with the change in
pH. The data suggests that the optimum binding of RNA/IRP complex occurred at pH 7.6.
Dissociation constant for two IRE RNA/IRP increased with an increase in ionic strength,
with a larger effect for FRT IRE RNA. This suggests that numerous electrostatic interactions
occur in the ferritin IRE RNA/IRP than ACO2 IRE RNA/IRP complex. Iodide quenching
shows that the majority of the tryptophan residues in IRP1 are solvent-accessible, assuming
that most of the tryptophan residues contribute to protein fluorescence.
Conclusion: The results obtained from this study clearly indicate that IRE RNA/IRP complex
is destabilized by the change in pH and ionic strength. These observations suggest that
both pH and ion are important for the assembly and stability of the IRE RNA/IRP complex