FIGURE 6.

Effect of magnesium on HPI. (A) Native gel electrophoresis on 10% polyacrylamide. HPI-wt (top) and HPI (bottom) were loaded in the buffer used for NMR studies with increasing magnesium concentration. The last lane (control) contains a thio-modified RNA at its 5′-end, which allows the visualization of a monomer (55-nt: S-RNA) and a dimer (110-nt: RNA-S-S-RNA). (B) Titration of HPI by magnesium by monitoring exchangeable protons. The assignment of imino protons based on the analysis of NOESY experiments is indicated for the Mg²⁺-free RNA (top) and the 10 mM Mg²⁺-RNA corresponding to a ratio of 57:1 (bottom). Broken lines and bold assignments indicate imino protons that undergo chemical shifts changes. (C) Magnesium titration of HPI using nonexchangeable protons. Overlays of regions of ¹H–¹³C HSQC spectra showing the aromatic H8-C8 of selectively labeled [¹³C/¹⁵N-G] HPI at 30°C, Mg²⁺-free (black) and upon successive additions of magnesium. Arrows indicate the cross-peaks that shifted upon addition of magnesium. (D) Summary of the effects observed in ¹H and ¹³C experiments on HPI upon Mg²⁺ binding. Small effects are indicated by one star, while stronger effects are highlighted using two stars.