Functional analysis of fractionated Drosophila Kc cell tRNA gene transcription components.
Functional analysis of fractionated Drosophila Kc cell tRNA gene transcription components.
复制标题
果蝇 Kc 细胞 tRNA 基因转录组分的功能分析。
DOI：
--
复制
发表时间：
1985
影响因子：
4.8
通讯作者：
D. Söll
中科院分区：
文献类型：
--
作者：
D. Burke;D. Söll
我们先前已经描述了从果蝇KC细胞提取物(Burke，D.J.，Schaack，J.，Sharp，S.和Söll，D.(1983)J.Biol)中部分纯化RNA聚合酶III转录成分。化学。258、15224-15231)。在这里，我们报道了被命名为因子B和C的两个转录所需组分的进一步的物理和功能分析。通过有序添加这些转录成分的稳定的络合物形成竞争实验，我们已经证明了因子C可以快速地与tRNA基因结合，但在没有因子B的情况下不会与DNA形成稳定的络合物。这些基因现在表现出果蝇系统的一个特性，即强烈依赖tRNA基因5‘侧翼区的序列。然而，KC因子B(凝胶过滤分析判断其表观分子量为260,000)与HeLa因子C不相容。这些结果，加上分离出包含所有必要转录成分的大复合体，强调了这些成分的相互作用对于稳定的复合体形成和转录启动的重要性。
We have previously described the partial purification of RNA polymerase III transcription components from Drosophila Kc cell extracts (Burke, D. J., Schaack, J., Sharp, S., and Söll, D. (1983) J. Biol. Chem. 258, 15224-15231). Here we report further physical and functional analysis of the two transcription-required fractions designated as factors B and C. Using stable complex formation-competition experiments with ordered addition of these transcription components, we have shown that factor C associates rapidly with the tRNA gene, but will not form a stable complex with the DNA in the absence of factor B. Reconstitution experiments of the individual Drosophila Kc cell factors with those from human HeLa cells demonstrated that the HeLa factor B, combined with Kc factor C, can form active transcription complexes. These now exhibit a property of the Drosophila system, a strong dependence on sequences in the 5'-flanking regions of tRNA genes. However, the Kc factor B (which has an apparent molecular weight of 260,000 as judged by gel filtration analysis) is not compatible with the HeLa factor C. These results, together with the isolation of a large complex containing all necessary transcription components, emphasize the importance of the interaction of these components for both stable complex formation and transcription initiation.