Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus
Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus
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DOI：
10.1073/pnas.1112934109
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发表时间：
2012-03-06
影响因子：
11.1
通讯作者：
Uchiumi, Toshio
中科院分区：
文献类型：
--
作者：
Nomura, Naoko;Honda, Takayoshi;Uchiumi, Toshio
核糖体上的蛋白质合成需要翻译GTPase因子以GTP结合的形式与核糖体结合，采取与GTP水解相结合的个体作用，并通常以gdp结合的形式解离。柔性核糖体柄蛋白的多个拷贝在这些过程中起重要作用。利用生化方法和来自超嗜热古细菌——阔氏焦球菌的茎蛋白，我们在这里提供了证据，证明了茎蛋白aP1的保守C端直接结合到延伸因子aEF-2的结构域I上，而不管aEF-2是与GTP还是GDP结合。定点突变表明，aP1 C端4个疏水氨基酸（Leu-100、103、106和ph -107）对直接结合至关重要。P1也被发现通过其C端与起始因子aIF5B以及aEF-1 α结合，但不与aIF2 γ结合。此外，分析性超离心和凝胶迁移分析表明，aP1和aP0的七聚体复合物aP0(aP1)(2)(aP1)(2)(aP1)(2) （aP1)(2)）可以同时结合多个aEF-2分子，这表明该因子可以接触到该茎蛋白的单个拷贝。利用真核蛋白P1/P2和P0也显示了茎蛋白C端的功能意义。古生菌和真核生物的茎秆蛋白的保守C端很可能在GTP水解前后都能与翻译因子结合。茎蛋白的这种一致的结合能力可能有助于维持核糖体周围高浓度的翻译因子，从而提高翻译效率。
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.