Assemblies of replication initiator protein on symmetric and asymmetric DNA sequences depend on multiple protein oligomerization surfaces.
Assemblies of replication initiator protein on symmetric and asymmetric DNA sequences depend on multiple protein oligomerization surfaces.
复制标题
复制起始蛋白在对称和不对称 DNA 序列上的组装取决于多个蛋白质寡聚表面。
DOI：
10.1006/jmbi.1998.2120
复制
发表时间：
1998
影响因子：
5.6
通讯作者：
Filutowicz,M
中科院分区：
文献类型：
--
作者：
Urh,M;Wu,J;Wu,J;Forest,K;Inman,RB;Filutowicz,M
质粒R6K的π 35.0蛋白通过结合一个DNA序列基序（TGAGR）来调节转录和复制，该基序在γ起始处不对称地排列成22 bp的正向重复序列（DR），或在其自身基因pir的操纵基因中对称地排列成反向半重复序列（IR）。π蛋白的两种天然形式及其异源二聚体的结合模式显示，主要种类π35.0（35.0 kDa）可以作为单体或二聚体结合DR的单个拷贝，而π30.5（30.5 kDa）仅作为二聚体结合。我们证明了π 35.0二聚体只有一个亚基与DNA特异性接触。电子显微镜（EM）分析由π 35.0和含有所有7个DR的DNA片段形成的核蛋白复合物，发现偶联（“手铐”）的DNA分子以平行方向排列。未观察到DNA的反平行取向。因此，手铐依赖于π 35.0在这样的结构中的高度有序的低聚。π蛋白（π35.0，π30.5）作为二聚体或异二聚体而不是单体与IR结合。此外，一个单一的氨基酸残基取代，F200 S（pir200），引入π 35.0，严重破坏了该蛋白在溶液中的二聚体的稳定性，并伴随着阻止该蛋白与IR的结合。这种突变也改变了π 35.0二聚体的稳定性，但它不改变π 35.0结合IR的能力。为了解释这些观察结果，我们提出π变体与DNA的不同相互作用是由蛋白质寡聚化的多个表面控制的。
The π35.0protein of plasmid R6K regulates transcription and replication by binding a DNA sequence motif (TGAGR) arranged either asymmetrically into 22 bp direct repeats (DRs) in the γ origin, or symmetrically into inverted half-repeats (IRs) in the operator of its own gene, pir. The binding patterns of the two natural forms of the π protein and their heterodimers revealed that the predominant species, π35.0(35.0 kDa), can bind to a single copy of the DR as either a monomer or a dimer while π30.5(30.5 kDa) binds only as a dimer. We demonstrate that only one subunit of a π35.0dimer makes specific contact with DNA. Electron microscopic (EM) analysis of the nucleoprotein complexes formed by π35.0and DNA fragments containing all seven DRs revealed coupled (“hand-cuffed”) DNA molecules that are aligned in a parallel orientation. Antiparallel orientations of the DNA were not observed. Thus, hand-cuffing depends on a highly ordered oligomerization of π35.0in such structures. The π protein (π35.0, π30.5) binds to an IR as a dimer or heterodimer but not as a monomer. Moreover, a single amino acid residue substitution, F200S (pir200), introduced into π30.5severely destabilizes dimers of this protein in solution and concomitantly prevents binding of this protein to the IR. This mutation also changes the stability of π35.0dimers but it does not change the ability of π35.0to bind IRs. To explain these observations we propose that the diverse interactions of π variants with DNA are controlled by multiple surfaces for protein oligomerization.