Nomura Takaomi, Nakatsuchi Masato, Sugita Daiyu, Nomura Mamoru, Kaminishi Tatsuya, Takemoto Chie, Shirouzu Mikako, Miyoshi Tomohiro, Yokoyama Shigeyuki, Hachimori Akira, Uchiumi Toshio
Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda 386-8567, Japan.
J Biochem. 2008 Nov;144(5):665-73. doi: 10.1093/jb/mvn113. Epub 2008 Sep 10.
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), while the recent X-ray crystallographic study and tandem MS analyses revealed the presence of a heptameric complex, L10(L12)(2), 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.
茎蛋白L12是50S核糖体亚基中唯一的多组分。在大肠杆菌中，两个L12二聚体与L10的C末端结构域结合形成五聚体复合物L10(L12)(2)，而最近的X射线晶体学研究和串联质谱分析表明，在一些嗜热细菌中存在七聚体复合物L10(L12)(2)。我们在此通过生化方法，利用嗜热栖热菌（Tt-）L10的C末端截短变体，对嗜热栖热菌L10和Tt-L12茎蛋白的复合物进行了表征。去除C末端的44个残基（Delta44）导致与Tt-L12的相互作用完全丧失。对组装在大肠杆菌50S核心颗粒上的Tt-L12与Tt-L10变体进行定量分析表明，野生型、Delta13和Delta23变体分别结合三个、两个和一个Tt-L12二聚体。含有嗜热栖热菌蛋白的杂交核糖体对大肠杆菌延伸因子高度可及。逐步去除Tt-L12二聚体会导致核糖体活性逐步降低，这表明每个单独的茎二聚体都对核糖体功能有贡献。有趣的是，即使只有两个或一个Tt-L12二聚体，杂交核糖体的EF-G依赖性GTPase活性也高于大肠杆菌核糖体。这一结果似乎归因于Tt-L12二聚体的结构特征。
