Imino proton NMR analysis of HDV ribozymes: nested double pseudoknot structure and Mg2+ ion-binding site close to the catalytic core in solution

Minimized trans-acting HDV ribozyme systems consisting of three (Rz-3) and two (Rz-2) RNA strands were prepared and their folding conformations were analyzed by NMR spectroscopy. The guanosine residues in one of the enzyme components of Rz-3 were labeled with ¹³C and ¹⁵N. Imino proton signals were assigned by analysis of NOESY and HSQC spectra. The results are consistent with the nested double pseudoknot model, which contains novel base pairs (P1.1), as observed in the crystal structure of a genomic HDV ribozyme. The NOE connectivities suggest an additional G:G pair at the bottom of P1.1 and at the top of P4. The effects of temperature and Mg²⁺ ions on base pairs for Rz-3 were examined. The temperature variation experiment on Rz-3 showed that P3 is the most stable and that P1.1 is as stable as P1 and P2. The imino proton signals of the G:U pair at the bottom of P1 and the top of P1.1, which are close to the cleavage site, showed the largest changes upon Mg²⁺ titration of Rz-3. The results suggest that the catalytic Mg²⁺ ion binds to the pocket formed by P1 and L3.