A zinc-binding site in the largest subunit of DNA-dependent RNA polymerase is involved in enzyme assembly
A zinc-binding site in the largest subunit of DNA-dependent RNA polymerase is involved in enzyme assembly
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DOI：
10.1101/gad.13.18.2439
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发表时间：
1999-09-15
影响因子：
10.5
通讯作者：
Severinov, K
中科院分区：
文献类型：
--
作者：
Markov, D;Naryshkina, T;Severinov, K
所有多亚基 DNA 依赖性 RNA 聚合酶 (RNAP) 都是锌金属酶，每个酶分子至少存在两个锌原子。参与锌结合的 RNAP 残基以及锌离子在转录机制或 RNAP 结构中的功能作用尚不清楚。在这里，我们在大肠杆菌 RNAP 最大亚基 β' 中找到了四个半胱氨酸残基，它们协调与酶紧密相关的两个锌离子之一。在缺乏锌的情况下，或者当突变阻止锌结合时，体外组装的 RNAP 保留了正确的亚基化学计量，但不起作用。我们证明锌作为分子伴侣，将变性的 β' 转化为紧凑的构象，从而与其他 RNAP 亚基有效地结合。配位锌的β'残基在真细菌和叶绿体中是保守的，但在真核生物和古细菌的同系物中不存在。因此，锌参与 RNAP 组装可能是真细菌型酶的独特特征。
All multisubunit DNA-dependent RNA polymerases (RNAP) are zinc metalloenzymes, and at least two zinc atoms are present per enzyme molecule. RNAP residues involved in zinc binding and the functional role of zinc ions in the transcription mechanism or RNAP structure are unknown. Here, we locate four cysteine residues in the Escherichia coli RNAP largest subunit, beta', that coordinate one of the two zinc ions tightly associated with the enzyme. In the absence of zinc, or when zinc binding is prevented by mutation, the in vitro-assembled RNAP retains the proper subunit stoichiometry but is not functional. We demonstrate that zinc acts as a molecular chaperone, converting denatured beta' into a compact conformation that productively associates with other RNAP subunits. The beta' residues coordinating zinc are conserved throughout eubacteria and chloroplasts, but are absent from homologs from eukaryotes and archaea. Thus, the involvement of zinc in the RNAP assembly may be a unique feature of eubacterial-type enzymes.