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首页> 外文期刊>Journal of Molecular Biology >FUNCTIONAL MAP OF THE ALPHA SUBUNIT OF ESCHERICHIA COLI RNA POLYMERASE - INSERTION ANALYSIS OF THE AMINO-TERMINAL ASSEMBLY DOMAIN
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FUNCTIONAL MAP OF THE ALPHA SUBUNIT OF ESCHERICHIA COLI RNA POLYMERASE - INSERTION ANALYSIS OF THE AMINO-TERMINAL ASSEMBLY DOMAIN

机译：大肠埃希氏菌RNA聚合酶α亚基的功能图-氨基末端组装域的插入分析

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摘要

The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme. Deletion analysis of alpha indicated that the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 is involved in this assembly. For fine mapping of the site(s) within this region required for subunit-subunit contacts, we constructed a set of insertion mutants of the rpoA gene, each encoding mutant alpha with two extra amino acid residues, Ala (A) and Ser (S), inserted at 20 residue intervals. The over-expressed alpha derivatives were purified to apparent homogeneity and examined for their ability to form dimers and to assemble beta and beta' subunits into core enzymes in vitro. Among a total of 11 alpha insertion derivatives tested, four mutants having the insertion at dispersed positions retained the ability to form active core enzymes. Other mutants showed defects in core enzyme assembly at various steps depending on the position of AS insertion: one mutant formed an unstable alpha(2) beta complex; one mutant exhibited decreased binding of beta' subunit; and five mutants did not form stable a dimers, of which one formed an alpha(2) beta complex and another formed an alpha beta complex. These results suggest that alpha dimerization involves multiple contact sites. Among alpha mutants with dimer formation ability, the mutation at amino acid residue 80 interfered with the binding of both beta and beta' subunits, and the mutation at position 200 made the alpha dimer inactive in beta' binding. [References: 21]
机译：大肠杆菌RNA聚合酶的α亚基在核心酶的组装中起关键作用。对α的缺失分析表明，由21位和235位之间的215个氨基酸残基组成的氨基末端结构域参与了该组装。为了精确定位亚单位-亚单位接触所需的该区域内的位点，我们构建了rpoA基因的插入突变体集，每个突变体编码带有两个额外氨基酸残基的Ala（A）和Ser（S ），以20个残基间隔插入。将过表达的α衍生物纯化至明显的同质性，并检查它们在体外形成二聚体以及将β和β'亚基组装成核心酶的能力。在总共测试的11种α插入衍生物中，在分散位置具有插入的四个突变体保留了形成活性核心酶的能力。其他突变体根据AS插入的位置，在各个步骤的核心酶组装过程中均显示出缺陷：一个突变体形成了不稳定的alpha（2）β复合物;一个突变体表现出减少的β'亚基结合;和五个突变体没有形成稳定的二聚体，其中一个形成了alpha（2）beta复合物，另一个形成了alpha beta复合物。这些结果表明，α二聚化涉及多个接触位点。在具有二聚体形成能力的α突变体中，氨基酸残基80处的突变干扰了β和β'亚基的结合，并且在200位的突变使α二聚体在β'结合中失活。 [参考：21]

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