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J. Biol. Chem., Vol. 282, Issue 45, 32827-32833, November 9, 2007

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Three Binding Sites for Stalk Protein Dimers Are Generally Present in Ribosomes from Archaeal Organism^*

Yasushi Maki¹, Tetsuo Hashimoto, Min Zhou^¶, Takao Naganuma, Jun Ohta, Takaomi Nomura^||, Carol V. Robinson^¶, and Toshio Uchiumi²

From the Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan, the Graduate School of Life and Environmental Science, University of Tsukuba, Ibaraki 305-8577, Japan, the ^¶Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom, and the ^||Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan

Ribosomes have a characteristic protuberance termed the stalk, which is indispensable for ribosomal function. The ribosomal stalk has long been believed to be a pentameric protein complex composed of two sets of protein dimers, L12-L12, bound to a single anchor protein, although ribosomes carrying three L12 dimers were recently discovered in a few thermophilic bacteria. Here we have characterized the stalk complex from Pyrococcus horikoshii, a thermophilic species of Archaea. This complex is known to be composed of proteins homologous to eukaryotic counterparts rather than bacterial ones. In truncation experiments of the C-terminal regions of the anchor protein Ph-P0, we surprisingly observed three Ph-L12 dimers bound to the C-terminal half of Ph-P0, and the binding site for the third dimer was unique to the archaeal homologs. The stoichiometry of the heptameric complex Ph-P0(Ph-L12)₂(Ph-L12)₂(Ph-L12)₂ was confirmed by mass spectrometry of the intact complex. In functional tests, ribosomes carrying a single Ph-L12 dimer had significant activity, but the addition of the second and third dimers increased the activity. A bioinformatics analysis revealed the evidence that ribosomes from all archaeal and also from many bacterial organisms may contain a heptameric complex at the stalk, whereas eukaryotic ribosomes seem to contain exclusively a pentameric stalk complex, thus modifying our view of the stalk structure significantly.

Received for publication, July 2, 2007 , and in revised form, August 30, 2007.

^* This work was supported by Grant-in-Aid for Scientific Research 14035222, a research grant from the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. This study was also supported by a grant for the promotion of Niigata University Research Projects. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5.

¹ Present address: Dept. of Physics, Osaka Medical College, Osaka 569-8686, Japan.

² To whom correspondence should be addressed: Dept. of Biology, Faculty of Science, Niigata University, Ikarashi 2-8050, Niigata 950-2181, Japan. Tel.: 81-25-262-7792; Fax: 81-25-262-7792; E-mail: uchiumi{at}bio.sc.niigata-u.ac.jp.

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