title: In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits.
abstract: We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0.Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0.Ph-L12 complex and Ph-L11 could replace L10.L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.
authors.affiliation: Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan.
authors.forename: Takaomi
authors.initials: T
authors.lastname: Nomura
authors.forename: Kohji
authors.initials: K
authors.lastname: Nakano
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Maki
authors.forename: Takao
authors.initials: T
authors.lastname: Naganuma
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakashima
authors.forename: Isao
authors.initials: I
authors.lastname: Tanaka
authors.forename: Makoto
authors.initials: M
authors.lastname: Kimura
authors.forename: Akira
authors.initials: A
authors.lastname: Hachimori
authors.forename: Toshio
authors.initials: T
authors.lastname: Uchiumi
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure; D020558:GTP Phosphohydrolases / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics; D043902:Pyrococcus horikoshii / Q000235:genetics*; D012335:RNA, Ribosomal / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D019843:Archaeal Proteins; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D012335:RNA, Ribosomal; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; C054675:ribosomal protein P0; D020558:GTP Phosphohydrolases