Modulation of conformational changes in helix 69 mutants by pseudouridine modifications

•Pseudouridine (Ψ) modulates pH-dependent structural changes of helix 69 (H69) RNAs.•Mutant A1912G requires Ψs for pH-dependent structural changes in H69 loop region.•A1919G mutation leads to formation of a U/Ψ1911•G1919 wobble pair.•G1919 and Ψ modifications work synergistically to modulate H69 structural changes.•U1917C mutation leads to pH-dependent changes in the H69 loop and stem regions.

Centrally located at the ribosomal subunit interface and mRNA tunnel, helix 69 (H69) from 23S rRNA participates in key steps of translation. Ribosome activity is influenced by three pseudouridine modifications, which modulate the structure and conformational behavior of H69. To understand how H69 is affected by the presence of pseudouridine in combination with sequence changes, the biophysical properties of wild-type H69 and representative mutants (A1912G, U1917C, and A1919G) were examined. Results from NMR and circular dichroism spectroscopy indicate that pH-dependent structural changes of wild-type H69 and the chosen mutants are modulated by pseudouridine and loop sequence. The effects of the mutations on global stability of H69 are negligible; however, pseudouridine stabilizes H69 at low pH conditions. Alterations to induced conformational changes of H69 likely result in compromised function, as indicated by previous biological studies.

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