Madin Kairat, Sawasaki Tatsuya, Kamura Nami, Takai Kazuyuki, Ogasawara Tomio, Yazaki Kazumori, Takei Toshiaki, Miura Kin-Ichiro, Endo Yaeta
Cell-free Science and Technology Research Center, and The Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.
FEBS Lett. 2004 Mar 26;562(1-3):155-9. doi: 10.1016/S0014-5793(04)00221-2.
We report a morphological study of functioning ribosomes in a efficient and robust cell-free protein synthesis system prepared from wheat embryos. Sucrose density gradient analysis of translated mixtures programmed with luciferase mRNAs having different 5' and 3' untranslated regions showed formation of large polysomes. Electron microscopic examination of translation mixtures programmed with those of capped and polyadenylated mRNA revealed that ribosomes assemble into a circular-type polysome in vitro. Furthermore, a series of experiments using mRNAs lacking either cap, poly(A) tail or both also resulted in the formation of circular polysomes, which are indistinguishable from those with the original mRNA. The wheat germ cell-free system may provide a good experimental system for understanding functional ribosomes at the molecular level.
我们报道了在由小麦胚制备的高效且稳定的无细胞蛋白质合成系统中对功能性核糖体的形态学研究。对用具有不同5'和3'非翻译区的荧光素酶mRNA编程的翻译混合物进行蔗糖密度梯度分析，结果显示形成了大型多核糖体。对用加帽和聚腺苷酸化mRNA编程的翻译混合物进行电子显微镜检查发现，核糖体在体外组装成环状多核糖体。此外，一系列使用缺乏帽、聚（A）尾或两者的mRNA的实验也导致了环状多核糖体的形成，这些环状多核糖体与用原始mRNA形成的多核糖体没有区别。小麦胚无细胞系统可能为在分子水平上理解功能性核糖体提供一个良好的实验系统。
