Laboratory of DNA Segregation and Life Cycle of Proteobacteria, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland.
Int J Mol Sci. 2022 Sep 1;23(17):9964. doi: 10.3390/ijms23179964.
The RA3 plasmid, the archetype of IncU incompatibility group, represents a mosaic-modular genome of 45.9 kb. The replication module encompasses and (initiator) surrounded by two long repetitive sequences DR1 and DR2 of unknown function. Here, we mapped the origin of replication to the 3' end of and showed that was activated by the transcription coming from in the adjacent stability module. Using various in vivo and in vitro methods we demonstrated that the expression proceeded either from located in the intergenic region or from the upstream strong that was autoregulated by RepA. Additionally, the activity was modulated by the transcription from the overlapping, divergently oriented . Both mRNA (antisense for mRNA) and its small polypeptide product, RepX, were strong incompatibility determinants. Hence, we showed that the sophisticated RA3 copy number control combined the multivalent regulation of expression, RepB titration by DR1, and transcriptional activation of , dependent on the RA3 global regulatory network. Similarly organized replicons have been found in diverse bacterial species confirming the significance of these mechanisms in establishing the IncU plasmids in a broad spectrum of hosts.
RA3 质粒是 IncU 不相容群的原型，代表了一个 45.9kb 的镶嵌模块基因组。复制模块包含 和 （启动子），周围是两个功能未知的长重复序列 DR1 和 DR2。在这里，我们将复制起点 映射到 的 3' 端，并表明 被来自相邻稳定模块中的 的转录激活。使用各种体内和体外方法，我们证明了 表达可以从位于基因间 区域的 或由 RepA 自身调控的上游强 进行。此外， 的活性受到来自重叠、反向转录的 的转录的调节。mRNA（mRNA 的反义）及其小多肽产物 RepX 都是强烈的不相容决定因素。因此，我们表明，复杂的 RA3 拷贝数控制结合了 表达的多价调节、DR1 对 RepB 的滴定以及 依赖于 RA3 全局调控网络的转录激活。在不同的细菌物种中发现了类似组织的复制子，这证实了这些机制在 IncU 质粒在广泛宿主中的建立中的重要性。
