Rutgers C A, Schaap P J, van 't Riet J, Woldringh C L, Raué H A
Biochemisch Labatorium, Vrije Universiteit, Amsterdam, The Netherlands.
Biochim Biophys Acta. 1990 Aug 27;1050(1-3):74-9. doi: 10.1016/0167-4781(90)90144-q.
We have developed a combination of in vivo and in vitro methods which allows us to determine the effect of practically every structural change, deletions as well as point mutations, on various biological functions of a ribosomal protein (r-protein). We have used this approach to delineate the functional domains of r-protein L25 from Saccharomyces cerevisiae. By analysis of the intracellular distribution of fusion proteins carrying various portions of L25 linked to Escherichia coli beta-galactosidase we traced the nuclear localization signal(s) of L25 to the region encompassing the N-terminal 61 amino acids of the protein. On the other hand, using in vitro prepared fragments of L25 we located the domain responsible for its specific binding to 26S rRNA to the region between amino acids 61 and 135. In order to be able to analyze the effect of mutations in L25 on ribosome biogenesis and function in vivo we constructed a mutant yeast strain in which the chromosomal L25 gene is placed under control of the inducible yeast GAL promoter. Since this strain is unable to grow on glucose as a carbon source the L25 gene must be essential for cell viability. Growth on glucose can be restored by introduction of a wild-type L25 gene on a plasmid, demonstrating that under these conditions the cells are dependent upon an extrachromosomally supplied copy of the gene.
我们开发了一套体内和体外方法的组合，这使我们能够确定几乎每一种结构变化，包括缺失和点突变，对核糖体蛋白（r蛋白）各种生物学功能的影响。我们已使用这种方法来描绘酿酒酵母r蛋白L25的功能结构域。通过分析携带与大肠杆菌β-半乳糖苷酶相连的L25不同部分的融合蛋白的细胞内分布，我们将L25的核定位信号追溯到该蛋白N端61个氨基酸的区域。另一方面，使用体外制备的L25片段，我们将其与26S rRNA特异性结合的结构域定位到氨基酸61至135之间的区域。为了能够在体内分析L25突变对核糖体生物合成和功能的影响，我们构建了一个突变酵母菌株，其中染色体L25基因置于可诱导的酵母GAL启动子的控制之下。由于该菌株不能以葡萄糖作为碳源生长，因此L25基因对细胞活力必不可少。通过在质粒上引入野生型L25基因可以恢复在葡萄糖上的生长，这表明在这些条件下细胞依赖于该基因的染色体外供应拷贝。
