Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
PLoS One. 2013 Apr 16;8(4):e61668. doi: 10.1371/journal.pone.0061668. Print 2013.
Plasmid pSW200 from Pantoea stewartii contains 41 copies of 15-bp repeats and has a replicon that is homologous to that of ColE1. Although deleting the repeats (pSW207) does not change the copy number and stability of the plasmid. The plasmid becomes unstable and is rapidly lost from the host when a homoplasmid with the repeats (pSW201) is present. Deleting the repeats is found to reduce the transcriptional activity of RNAIp and RNAIIp by about 30%, indicating that the repeats promote the transcription of RNAI and RNAII, and how the RNAI that is synthesized by pSW201 inhibits the replication of pSW207. The immunoblot analysis herein demonstrates that RNA polymerase β subunit and σ(70) in the lysate from Escherichia coli MG1655 bind to a biotin-labeled DNA probe that contains the entire sequence of the repeat region. Electrophoretic mobility shift assay also reveals that purified RNA polymerase shifts a DNA probe that contains four copies of the repeats. These results thus obtained reveal that RNA polymerase holoenzyme binds to the repeats. The repeats also exchange RNA polymerase with RNAIp and RNAIIp in vitro, revealing the mechanism by which the transcription is promoted. This investigation elucidates a mechanism by which a plasmid prevents the invasion of an incompatible plasmid and maintains its stability in the host cell during evolution.
来自 Pantoea stewartii 的质粒 pSW200 含有 41 个 15 碱基重复序列，其复制子与 ColE1 的复制子同源。尽管删除重复序列（pSW207）不会改变质粒的拷贝数和稳定性，但当存在带有重复序列的同质粒（pSW201）时，质粒变得不稳定并迅速从宿主中丢失。研究发现，删除重复序列会使 RNAIp 和 RNAIIp 的转录活性降低约 30%，表明重复序列促进了 RNAI 和 RNAII 的转录，以及合成的 RNAI 如何抑制 pSW207 的复制。本文的免疫印迹分析表明，来自大肠杆菌 MG1655 的裂解物中的 RNA 聚合酶 β 亚基和 σ(70)与包含重复区域整个序列的生物素标记 DNA 探针结合。电泳迁移率变动分析也表明，纯化的 RNA 聚合酶使包含四个重复的 DNA 探针发生迁移。这些结果表明 RNA 聚合酶全酶与重复序列结合。重复序列还在体外与 RNAIp 和 RNAIIp 交换 RNA 聚合酶，揭示了转录被促进的机制。该研究阐明了一种质粒防止不相容质粒入侵并在进化过程中在宿主细胞中保持其稳定性的机制。
