Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus

被引:46
作者
Nomura, Naoko [1 ]
Honda, Takayoshi [2 ]
Baba, Kentaro [2 ]
Naganuma, Takao [1 ,2 ]
Tanzawa, Takehito [1 ]
Arisaka, Fumio [3 ]
Noda, Masanori [4 ]
Uchiyama, Susumu [4 ]
Tanaka, Isao [1 ]
Yao, Min [1 ]
Uchiumi, Toshio [2 ]
机构
[1] Hokkaido Univ, Fac Adv Life Sci, Kita Ku, Sapporo, Hokkaido 0600810, Japan
[2] Niigata Univ, Fac Sci, Dept Biol, Nishi Ku, Niigata 9502181, Japan
[3] Tokyo Inst Technol, Grad Sch Biosci & Biotechnol, Midori Ku, Yokohama, Kanagawa 2268501, Japan
[4] Osaka Univ, Grad Sch Engn, Dept Biotechnol, Suita, Osaka 5650871, Japan
关键词
GTPase-associated center; ribosome protein P0; ribosome protein P1; hyperthermophilic archaeon; ELONGATION-FACTOR-G; GTPASE-ASSOCIATED CENTER; EF-TU; STRUCTURAL BASIS; BINDING; P0; P1; P2; ACTIVATION; DOMAIN;
D O I
10.1073/pnas.1112934109
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Protein synthesis on the ribosome requires translational GTPase factors to bind to the ribosome in the GTP-bound form, take individual actions that are coupled with GTP hydrolysis, and dissociate, usually in the GDP-bound form. The multiple copies of the flexible ribosomal stalk protein play an important role in these processes. Using biochemical approaches and the stalk protein from a hyperthermophilic archaeon, Pyrococcus horikoshii, we here provide evidence that the conserved C terminus of the stalk protein aP1 binds directly to domain I of the elongation factor aEF-2, irrespective of whether aEF-2 is bound to GTP or GDP. Site-directed mutagenesis revealed that four hydrophobic amino acids at the C terminus of aP1, Leu-100, 103, 106, and Phe-107, are crucial for the direct binding. P1 was also found to bind to the initiation factor aIF5B, as well as aEF-1 alpha, but not aIF2 gamma, via its C terminus. Moreover, analytical ultracentrifugation and gel mobility shift analyses showed that a heptameric complex of aP1 and aP0, aP0(aP1)(2)(aP1)(2)(aP1)(2), can bind multiple aEF-2 molecules simultaneously, which suggests that individual copies of the stalk protein are accessible to the factor. The functional significance of the C terminus of the stalk protein was also shown using the eukaryotic proteins P1/P2 and P0. It is likely that the conserved C terminus of the stalk proteins of archaea and eukaryotes can bind to translation factors both before and after GTP hydrolysis. This consistent binding ability of the stalk protein may contribute to maintaining high concentrations of translation factors around the ribosome, thus promoting translational efficiency.
引用
收藏
页码:3748 / 3753
页数:6
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