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首页> 外文期刊>Nucleic Acids Research >Involvement of multiple subunit-subunit contacts in the assembly of RNA polymerase II.
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Involvement of multiple subunit-subunit contacts in the assembly of RNA polymerase II.

机译：RNA聚合酶II的组装中涉及多个亚基-亚基接触。

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摘要

RNA polymerase II from the fission yeast Schizo-saccharomyces pombe consists of 12 species of subunits, Rpb1-Rpb12. We expressed these subunits, except Rpb4, simultaneously in cultured insect cells with baculovirus expression vectors. For the isolation of subunit complexes formed in the virus-infected cells, a glutathione S -transferase (GST) sequence was fused to the rpb3 cDNA to produce GST-Rpb3 fusion protein and a decahistidine-tag sequence was inserted into the rpb1 cDNA to produce Rpb1H protein. After successive affinity chromatography on glutathione and Ni(2+)columns, complexes consisting of the seven subunits, Rpb1H, Rpb2, GST-Rpb3, Rpb5, Rpb7, Rpb8 and Rpb11, were identified. Omission of the GST-Rpb3 expression resulted in reduced assembly of the Rpb11 into the complex. Direct interaction between Rpb3 and the other six subunits was detected by pairwise coexpression experiments. Coexpression of various combinations of a few subunits revealed that Rpb11 enhances Rpb3-Rpb8 interaction and consequently Rpb8 enhances Rpb1-Rpb3 interaction to some extent. We propose a mechanism in which the assembly of RNA poly-merase II is stabilized through multiple subunit-subunit contacts.
机译：来自裂殖酵母裂殖酵母的RNA聚合酶II由12种亚基Rpb1-Rpb12组成。我们用杆状病毒表达载体在培养的昆虫细胞中同时表达了除Rpb4以外的这些亚基。为了分离在病毒感染的细胞中形成的亚基复合物，将谷胱甘肽S-转移酶（GST）序列与rpb3 cDNA融合以产生GST-Rpb3融合蛋白，并在cab- rpb1 cDNA中插入一个十氨基酸标签序列以产生Rpb1H蛋白。在谷胱甘肽和Ni（2+）柱上进行连续亲和层析后，鉴定出由七个亚基Rpb1H，Rpb2，GST-Rpb3，Rpb5，Rpb7，Rpb8和Rpb11组成的复合物。省略GST-Rpb3表达导致Rpb11组装成复合物的减少。通过成对共表达实验检测到Rpb3与其他六个亚基之间的直接相互作用。几个亚基的各种组合的共表达显示Rpb11增强Rpb3-Rpb8相互作用，因此Rpb8在一定程度上增强Rpb1-Rpb3相互作用。我们提出了一种机制，其中RNA聚合酶II的组装通过多个亚基-亚基接触得以稳定。

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