Helix 69 is a region of significant interest, which is located in the 50S subunit at the subunit interface of the ribosome. This research focused on identifying structural changes in this region upon changes in solution conditions such as pH and Mg 2+ concentration. Furthermore, the roles of naturally occurring, highly conserved modified nucleotides in helix 69, namely pseudouridine and 3-methylpseudouridine, were examined. Significant changes in structure and thermodynamic stability of helix 69 were observed due to changes in pH, which may play an important role in the biological functions of the region during certain stages of protein translation. Our experiments reveal pH and Mg 2+ -dependent base flipping of loop reduces in helix 69, as well as reduced thermodynamic stability of helix 69 caused by nucleotide (pseudouridine to uridine or 1-methylpseudouridine) or phosphate (to phosphorothioate) substitution. Highly conserved pseudouridines at three positions, 1911, 1915, and 1917, of helix 69 were found to be important for the pH-dependent structural changes in the region, as well as Mg 2+ binding. Cooperative structural and thermodynamic effects of pseudouridines in helix 69 were also examined. Significant cooperative structural and thermodynamic stability was observed for pseudouridines at positions 1911-1917 and 1915-1917. These studies provide information that will be useful in the design of new ribosome (specifically helix 69) targeting compounds as potential drug therapeutics.
