title: Pseudouridylation of 23S rRNA helix 69 promotes peptide release by release factor RF2 but not by release factor RF1.
abstract: Pseudouridine [Ψ] is a frequent base modification in the ribosomal RNA [rRNA] and may be involved in the modulation of the conformational flexibility of rRNA helix-loop structures during protein synthesis. Helix 69 of 23S rRNA contains pseudouridines at the positions 1911, 1915 and 1917 which are formed by the helix 69-specific synthase RluD. The growth defect caused by the lack of RluD can be rescued by mutations in class I release factor RF2, indicating a role for helix 69 pseudouridines in translation termination. We investigated the role of helix 69 pseudouridines in peptide release by release factors RF1 and RF2 in an in vitro system consisting of purified components of the Escherichia coli translation apparatus. Lack of all three pseudouridines in helix 69 compromised the activity of RF2 about 3-fold but did not significantly affect the activity of RF1. Reintroduction of pseudouridines into helix 69 by RluD-treatment restored the activity of RF2 in peptide release. A Ψ-to-C substitution at the 1917 position caused an increase in the dissociation rate of RF1 and RF2 from the postrelease ribosome. Our results indicate that the presence of all three pseudouridines in helix 69 stimulates peptide release by RF2 but has little effect on the activity of RF1. The interactions around the pseudouridine at the 1917 position appear to be most critical for a proper interaction of helix 69 with release factors.
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia.
authors.forename: Kalle
authors.initials: K
authors.lastname: Kipper
authors.forename: Sulev
authors.initials: S
authors.lastname: Sild
authors.forename: Csaba
authors.initials: C
authors.lastname: Hetényi
authors.forename: Jaanus
authors.initials: J
authors.lastname: Remme
authors.forename: Aivar
authors.initials: A
authors.lastname: Liiv
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances / Q000378:metabolism*; D008745:Methylation; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D010454:Peptide Termination Factors / Q000378:metabolism*; D017354:Point Mutation; D014176:Protein Biosynthesis; D011560:Pseudouridine / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D010454:Peptide Termination Factors; D012338:RNA, Ribosomal, 23S; C489005:prfA protein, E coli; C489006:prfB protein, E coli; D011560:Pseudouridine