Transcriptional Interference by a Complex Formed at the Centromere-Like Partition Site of Plasmid P1

The partition site, parS, promotes accurate segregation of the replicated P1 plasmid to daughter cells when the P1-encoded ParA and ParB proteins are supplied. The parS site was inserted into the Escherichia coli chromosome between the promoter and the structural gene for β-galactosidase, lacZ. There was little interference with lacZ expression when ParA and ParB were supplied in trans. However, when a mutant ParA protein, ParAM314I, was supplied along with ParB, expression of lacZ was shut down. ParAM314I, ParB, and parS appear to form a nucleoprotein complex that blocks transcription. Mutations in parA and parB that relieved the parAM314I-dependent block were found. In addition, new mutations which impose the block were selected. Five of the latter mapped to parA and one to parB; all had a propagation-defective phenotype (Par(PD)) similar to that of parAM314I. Thus, whereas a null par mutant P1 plasmid segregates its DNA randomly, these mutants prevent even random distribution of the plasmid. We propose that ParA protein normally interacts transiently with the ParB-parS complex for partition to proceed but that the mutations block ParA dissociation. This “permanent” ParA-ParB-parS complex acts as a transcription block. Consistent with this hypothesis, we found that three of the seven blocking mutations lie within regions of ParA and ParB that are known to interact with each other. When the transcription block is imposed, regional silencing of nearby genes occurs. However, the requirement for ParA and a mutant parA or parB allele distinguishes the transcription block from the regional ParB-dependent gene silencing previously described.

分隔位点parS可在提供P1编码的ParA与ParB蛋白时，促进复制后的P1质粒精准分离至子代细胞。将parS插入大肠杆菌（Escherichia coli）的染色体中，位于启动子（promoter）与β-半乳糖苷酶（β-galactosidase）的结构基因lacZ之间。当以反式方式提供ParA和ParB时，对lacZ的表达几乎无干扰。然而，当将突变型ParA蛋白ParAM314I与ParB共同提供时，lacZ的表达被完全抑制。ParAM314I、ParB与parS似乎可形成阻断转录的核蛋白复合物。研究发现了parA和parB中能够解除ParAM314I依赖性阻断的突变。此外，还筛选到了可引发该阻断效应的新突变。其中5个新突变定位至parA，1个定位至parB；所有这些突变均表现出与parAM314I相似的传播缺陷表型（Par(PD)）。因此，尽管无效par突变型P1质粒的DNA会随机分离，但这些突变体甚至会阻止质粒的随机分布。我们提出，正常情况下ParA蛋白会与ParB-parS复合物短暂相互作用，以完成质粒分离过程，而这些突变会阻断ParA的解离。这种“永久性”的ParA-ParB-parS复合物发挥转录阻断作用。与该假说一致，我们发现7个阻断突变中有3个位于已知可相互作用的ParA与ParB区域内。当施加转录阻断时，会发生邻近基因的区域沉默效应。不过，该转录阻断需要ParA以及突变型parA或parB等位基因的参与，这一点使其与此前报道的、依赖ParB的区域基因沉默效应区分开来。