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

Takaomi Nomura, Kohji Nakano, Yasushi Maki, Takao Naganuma, Takashi Nakashima, Isao Tanaka, Makoto Kimura, Akira Hachimori, Toshio Uchiumi

Research output: Contribution to journalArticle

25 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)565-571
Number of pages7
JournalBiochemical Journal
Volume396
Issue number3
DOIs
Publication statusPublished - Jun 15 2006

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Pyrococcus horikoshii
GTP Phosphohydrolases
Ribosomes
Escherichia coli
L 012
Peptide Elongation Factors
Proteins
Ribosomal Proteins
Gene encoding
In Vitro Techniques
Archaea
Mutant Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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. / Nomura, Takaomi; Nakano, Kohji; Maki, Yasushi; Naganuma, Takao; Nakashima, Takashi; Tanaka, Isao; Kimura, Makoto; Hachimori, Akira; Uchiumi, Toshio.

In: Biochemical Journal, Vol. 396, No. 3, 15.06.2006, p. 565-571.

Research output: Contribution to journalArticle

Nomura, Takaomi ; Nakano, Kohji ; Maki, Yasushi ; Naganuma, Takao ; Nakashima, Takashi ; Tanaka, Isao ; Kimura, Makoto ; Hachimori, Akira ; Uchiumi, Toshio. / 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. In: Biochemical Journal. 2006 ; Vol. 396, No. 3. pp. 565-571.
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