Röhl R, Nierhaus K H
Proc Natl Acad Sci U S A. 1982 Feb;79(3):729-33. doi: 10.1073/pnas.79.3.729.
Stoichiometric amounts of ribosomal proteins and RNA derived from the 50S subunit reconstitute to fully active particles under the conditions of a two-step incubation procedure. After the first incubation, all components are found in a particle that is activated in the second incubation [Dohme, F. & Nierhaus, K. H. (1976) J. Mol. Biol. 107, 585-599]. Here we describe the assembly dependences of the ribosomal components in the first incubation. Assembly dependence is the requirements of one protein that, before it binds, another must be first built into the ribosome. After incubation of 23S RNA and the proteins under observation, the mixture was subjected to sucrose gradient analysis. The RNA-protein complex was precipitated with trichloroacetic acid and the proteins were identified by NaDodSO4 gel electrophoresis. The assembly dependences of 26 proteins could be elucidated. In a second series of experiments, the incorporation of 3H-labeled 5S RNA in the 23S-protein complex was analyzed. It was found that L5, L15, and L18 are absolutely required for 5S RNA incorporation. In addition, two of the three proteins L2, L3, and L4 are needed, in excellent agreement with the protein dependences. The data are summarized in an assembly map. Comparison with other data shows a structural domain at the 5' end of 23S RNA around protein L20 combining all proteins essential in the early assembly. All the proteins essential for the reconstitution of the peptidyltransferase protein form a skeleton of strong assembly dependences. Finally, L proteins whose genes are present in large transcriptional units on the chromosome depend on each other during assembly.
在两步温育程序的条件下，化学计量的源自50S亚基的核糖体蛋白和RNA重新组装成完全有活性的颗粒。第一次温育后，所有组分都存在于一个在第二次温育中被激活的颗粒中[多姆，F. & 尼尔豪斯，K. H.（1976年）《分子生物学杂志》107卷，585 - 599页]。在此，我们描述第一次温育中核糖体组分的组装依赖性。组装依赖性是指一种蛋白质在结合之前，另一种蛋白质必须先组装到核糖体中。将23S RNA和所观察的蛋白质温育后，混合物进行蔗糖梯度分析。RNA - 蛋白质复合物用三氯乙酸沉淀，蛋白质通过十二烷基硫酸钠凝胶电泳鉴定。可以阐明26种蛋白质的组装依赖性。在第二系列实验中，分析了3H标记的5S RNA掺入23S - 蛋白质复合物的情况。发现L5、L15和L18是5S RNA掺入绝对必需的。此外，L2、L3和L4这三种蛋白质中的两种是必需的，这与蛋白质依赖性非常一致。数据总结在一个组装图中。与其他数据比较显示，在23S RNA 5'端围绕蛋白质L20处有一个结构域，该结构域结合了早期组装中所有必需的蛋白质。肽基转移酶蛋白重建所需的所有蛋白质形成了一个具有强组装依赖性的骨架。最后，其基因存在于染色体上大转录单位中的L蛋白在组装过程中相互依赖。
