title: Hydroxylated histidine of human ribosomal protein uL2 is involved in maintaining the local structure of 28S rRNA in the ribosomal peptidyl transferase center.
abstract: Protein uL2 is essential for the catalytic activity of the ribosome and has a conserved shape in ribosomes from all domains of life. However, the sequence of its unstructured C-terminal loop apex that contacts the conserved 23S/28S rRNA helix (H) 93 near the ribosomal peptidyl transferase center differs in bacteria, archaea and eukaryotes. Eukaryote-specific residue His216 located in this loop in mammalian uL2 is hydroxylated in ribosomes. We used a set of chemical probes to explore the structure of an RNA that mimicked a segment of 28S rRNA domain V containing part of the uL2 binding site including H93, complexed with either natural (hydroxylated) or recombinant (unmodified) human uL2. It was found that both protein forms engage H93 during binding, but only natural uL2 (uL2n) protects it from hydroxyl radicals. The association of uL2n with RNA leads to changes in its structure at U4532 adjacent to the universally conserved U4531 (U2585, Escherichia coli numbering) involved in peptidyl transferase center formation, and at the universally conserved C4447 (2501) located in the ribosome near A4397 (2451) and C3909 (2063) belonging to the peptidyl transferase center. As a result, both nucleotides become strongly exposed to hydroxyl radicals. Our data argue that the hydroxyl group at His216 in the C-terminal loop apex of mammalian uL2 contributes to stabilization of a protein conformation that is favorable for binding to H93 of 28S rRNA and that this binding induces structural rearrangement in the regions close to the peptidyl transferase center in the mature ribosome.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia.
authors.forename: Darya D
authors.initials: DD
authors.lastname: Yanshina
authors.forename: Konstantin N
authors.initials: KN
authors.lastname: Bulygin
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Malygin
authors.forename: Galina G
authors.initials: GG
authors.lastname: Karpova
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D005260:Female; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006900:Hydroxylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010458:Peptidyl Transferases / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; D006639:Histidine; D010458:Peptidyl Transferases
keywords: RNA footprinting; mammalian ribosome structure; peptidyl transferase center; protein hydroxylation; ribosomal protein uL2