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Journal of Biochemistry Advance Access originally published online on September 10, 2008
Journal of Biochemistry 2008 144(5):665-673; doi:10.1093/jb/mvn113
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© 2008 The Japanese Biochemical Society

Biochemical Evidence for the Heptameric Complex L10(L12)6 in the Thermus thermophilus Ribosome: In Vitro Analysis of its Molecular Assembly and Functional Properties

Takaomi Nomura1, Masato Nakatsuchi1, Daiyu Sugita1, Mamoru Nomura2,3, Tatsuya Kaminishi2,4, Chie Takemoto2, Mikako Shirouzu2, Tomohiro Miyoshi5, Shigeyuki Yokoyama2,6, Akira Hachimori1 and Toshio Uchiumi5,*

1Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda 386-8567; 2Systems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045; 3Shimoda Marine Research Center, University of Tsukuba, 5-10-1 Shimoda, Shizuoka 415-0025, Japan; 4Cluster of Excellence for Macromolecular Complexes, Institut für Organische Chemie und Chemische Biologie, J.W. Goethe-Universitæt, Frankfurt am Main, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany; 5Department of Biology, Faculty of Science, Niigata University, 2-8050 Ikarashi, Niigata 950-2181; and 6Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

*To whom correspondence should be addressed. Tel/Fax: +81-25-262-7792, E-mail: uchiumi{at}bio.sc.niigata-u.ac.jp

Received July 10, 2008; Accepted September 5, 2008


  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 ({Delta}44) 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, {Delta}13 and {Delta}23 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.

Key Words: elongation factors, GTPase-associated centre, ribosomal stalk, ribosome, translation elongation

Abbreviations: GST, glutathione S-transferase; WT, wild-type


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T. Miyoshi, T. Nomura, and T. Uchiumi
Engineering and Characterization of the Ribosomal L10{middle dot}L12 Stalk Complex: A STRUCTURAL ELEMENT RESPONSIBLE FOR HIGH TURNOVER OF THE ELONGATION FACTOR G-DEPENDENT GTPase
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[Abstract] [Full Text] [PDF]


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