Inada Toshifumi, Winstall Eric, Tarun Salvador Z, Yates John R, Schieltz Dave, Sachs Alan B
Department of Molecular and Cell Biology, University of California at Berkeley, 94720, USA.
RNA. 2002 Jul;8(7):948-58. doi: 10.1017/s1355838202026018.
We describe a one-step affinity method for purifying ribosomes from the budding yeast Saccharomyces cerevisiae. Extracts from yeast strains expressing only C-terminally tagged Rpl25 protein or overexpressing this protein in the presence of endogenous Rpl25p were used as the starling materials. The purification was specific for tagged 60S subunits, and resulted in the copurification of 80S subunits and polysomes, as well as ribosome-associated proteins and mRNAs. Two of these associated proteins, Mpt4p and Asc1p, were nearly stoichiometrically bound to the ribosome. In addition, the degree of mRNA association with the purified ribosomes was found to reflect the mRNA's translational status within the cell. The one-step purification of ribosome and its associated components from a crude extract should provide an important tool for future structural and biochemical studies of the ribosome, as well as for expression profiling of translated mRNAs.
我们描述了一种从出芽酵母酿酒酵母中纯化核糖体的一步亲和方法。仅表达C末端标记的Rpl25蛋白的酵母菌株提取物或在内源Rpl25p存在下过表达该蛋白的酵母菌株提取物用作起始材料。该纯化方法对标记的60S亚基具有特异性，并导致80S亚基和多核糖体以及核糖体相关蛋白和mRNA的共纯化。其中两种相关蛋白Mpt4p和Asc1p与核糖体的结合接近化学计量。此外，发现与纯化核糖体结合的mRNA程度反映了该mRNA在细胞内的翻译状态。从粗提物中一步纯化核糖体及其相关成分，应为核糖体未来的结构和生化研究以及翻译mRNA的表达谱分析提供重要工具。
