Pseudouridylation of 23S rRNA helix 69 promotes peptide release by release factor RF2 but not by release factor RF1

► Depletion of Ψ-s from helix 69 impairs RF2-dependent peptide release. ► Reintroduction of Ψ-s into helix 69 stimulates RF2-dependent peptide release. ► RF1-dependent peptide release is not dependent on Ψ-s in helix 69. ► Interactions around Ψ1917 are important for RF-helix 69 interactions.