REPLICATIVELY ACTIVE COMPLEXES OF DNAA PROTEIN AND THE ESCHERICHIA-COLI CHROMOSOMAL ORIGIN OBSERVED IN THE ELECTRON-MICROSCOPE
REPLICATIVELY ACTIVE COMPLEXES OF DNAA PROTEIN AND THE ESCHERICHIA-COLI CHROMOSOMAL ORIGIN OBSERVED IN THE ELECTRON-MICROSCOPE
复制标题
DOI：
10.1006/jmbi.1993.1481
复制
发表时间：
1993-09-05
影响因子：
5.6
通讯作者：
KORNBERG, A
中科院分区：
文献类型：
--
作者：
CROOKE, E;THRESHER, R;KORNBERG, A
在DNA复制的早期阶段，Dna A蛋白和大肠杆菌原核染色体起源(ORIC)形成了一个初始复合体。我们使用电子显微镜来确定在这个多组分系统的重建过程中形成的几个结构中，哪个结构是复制活性络合物。一种独特的结构可能与活性和局部化的TooriC相关，而其他几种结构则不能。在下一个起始阶段形成开放的复合体，伴随着一个在大小和形状上与功能初始复合体相似的结构的存在。虽然最初的复合体与ATP或DNAA蛋白的ADP形式都可以观察到，但只有ATP型蛋白能有效地产生开放的复合体。通过对已知对复制很重要的几个DNA序列元件的诱变来确定这些改变对初始复合体形成的影响。根据电子显微镜和功能分析的判断，包含四个9-merdna Abox的区域被证明是形成初始复合体所必需的，而三个连续的富含AT的13-MERS，即已知的开放OforiC的位置，则不是。
DnaA protein and theEscherichia colichromosomal origin (oriC) form an initial complex at an early stage in the initiation of DNA replication. We have used electron microscopy to determine which structure among the several formed in the reconstitution of this multicomponent system is the replicatively active complex. One distinctive structure could be correlated with activity and localized tooriC, whilst several others could not. Formation of an open complex in the next stage of initiation was accompanied by the presence of a structure similar in size and shape to that of the functional initial complex. Whereas the initial complex was observed with either ATP or the ADP-forms of DnaA protein, only the ATP-form was effective in producing the open complex. Mutagenesis of several DNA sequence elements inoriC, known to be important for replication, was employed to determine the effects of these alterations on formation of the initial complex. As judged by electron microscopy and by functional assays, the region containing the four 9-merdnaAboxes proved to be essential for the formation of the initial complex, while the three contiguous AT-rich 13-mers, known sites for opening oforiC, were not.