One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs
One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs
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DOI：
10.1017/s1355838202026018
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发表时间：
2002-07-01
期刊：
影响因子：
4.5
通讯作者：
Sachs, AB
中科院分区：
文献类型：
--
作者：
Inada, T;Winstall, E;Sachs, AB
我们描述了一个一步亲和法纯化核糖体从芽殖酵母酿酒酵母。将来自仅表达C-末端标记的Rp 125蛋白或在内源性Rp 125 p存在下过表达该蛋白的酵母菌株的提取物用作起始材料。该纯化对标记的60 S亚基具有特异性，并导致80 S亚基和多聚核糖体以及核糖体相关蛋白和mRNA的共纯化。其中两个相关蛋白质，Mpt 4p和Asc 1 p，几乎化学计量结合的核糖体。此外，发现mRNA与纯化的核糖体的结合程度反映了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组分将为核糖体的未来结构和生物化学研究以及翻译mRNA的表达谱分析提供重要工具。
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 Rp125 protein or overexpressing this protein in the presence of endogenous Rp125p were used as the starting 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.