title: Biochemical evidence for the heptameric complex L10(L12)6 in the Thermus thermophilus ribosome: in vitro analysis of its molecular assembly and functional properties.
abstract: The stalk protein L12 is the only multiple component in 50S ribosomal subunit. In Escherichia coli, two L12 dimers bind to the C-terminal domain of L10 to form a pentameric complex, L10[(L12)(2)](2), while the recent X-ray crystallographic study and tandem MS analyses revealed the presence of a heptameric complex, L10[(L12)(2)](3), in some thermophilic bacteria. We here characterized the complex of Thermus thermophilus (Tt-) L10 and Tt-L12 stalk proteins by biochemical approaches using C-terminally truncated variants of Tt-L10. The C-terminal 44-residues removal (Delta44) resulted in complete loss of interactions with Tt-L12. Quantitative analysis of Tt-L12 assembled onto E. coli 50S core particles, together with Tt-L10 variants, indicated that the wild-type, Delta13 and Delta23 variants bound three, two and one Tt-L12 dimers, respectively. The hybrid ribosomes that contained the T. thermophilus proteins were highly accessible to E. coli elongation factors. The progressive removal of Tt-L12 dimers caused a stepwise reduction of ribosomal activities, which suggested that each individual stalk dimer contributed to ribosomal function. Interestingly, the hybrid ribosomes showed higher EF-G-dependent GTPase activity than E. coli ribosomes, even when two or one Tt-L12 dimer. This result seems to be due to a structural characteristic of Tt-L12 dimer.
authors.affiliation: Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda 386-8567, Japan.
authors.forename: Takaomi
authors.initials: T
authors.lastname: Nomura
authors.forename: Masato
authors.initials: M
authors.lastname: Nakatsuchi
authors.forename: Daiyu
authors.initials: D
authors.lastname: Sugita
authors.forename: Mamoru
authors.initials: M
authors.lastname: Nomura
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Kaminishi
authors.forename: Chie
authors.initials: C
authors.lastname: Takemoto
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Miyoshi
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Akira
authors.initials: A
authors.lastname: Hachimori
authors.forename: Toshio
authors.initials: T
authors.lastname: Uchiumi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins