Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents.
Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents.
复制标题
大肠杆菌 RNA 聚合酶的亚基形貌。
作者：
Z. Hillel;C. Wu
用可裂解的双功能试剂4-巯基丁酰亚胺酸甲酯和不可裂解的试剂辛二酰亚胺酸二甲酯和N，N ′-（1，4-亚苯基）双马来酰亚胺，通过化学交联研究了大肠杆菌DNA依赖的RNA聚合酶全酶（α 2 β β ' sigma）和核心酶（α 2 β β'）的四级结构。从十二烷基硫酸盐-聚丙烯酰胺凝胶电泳鉴定的交联亚基邻居推导出的亚基组织模型表明，大的β和β '亚基构成核心和全酶的骨架，而σ和两个α亚基沿着β和β'亚基的接触结构域与该结构相互作用。在全酶中，σ亚基位于至少一个α亚基附近。这两个α亚基在全酶、核心酶和分离的α 2 β复合物中彼此接近。交联的“过早”的核心和全酶中间体在体外重建的活性酶从分离的亚基表明，这些物种是由亚基复合物的分子量低于天然核心和全酶，分别。RNA聚合酶及其亚复合物的结构信息对于酶-启动子识别以及酶的亚基组装机制具有重要意义。
The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holenzyme (alpha 2 beta beta' sigma) and core enzyme (alpha 2 beta beta') have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N'-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large beta and beta' subunits constitute the backbone of both core and holoenzyme, while sigma and two alpha subunits interact with this structure along the contact domain of beta and beta' subunits. In holoenzyme, sigma subunit is in the vicinity of at least one alpha subunit. The two alpha subunits are close to each other in holoenzyme, core enzyme, and the isolated alpha 2 beta complex. Cross-linking of the "premature" core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition as well as the mechanism of subunit assembly of the enzyme.