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首页> 外文期刊>Journal of Molecular Biology >Replication control of a small cryptic plasmid of Escherichia coli.
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Replication control of a small cryptic plasmid of Escherichia coli.

机译：大肠杆菌小密码质粒的复制控制。

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摘要

The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n'-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained. Copyright 1999 Academic Press.
机译：阐明了RepA起始蛋白在大肠杆菌小密码质粒pKL1的复制和拷贝数控制中的作用。鉴定出的ori区域包含拷贝数控制元件（cop）和活性单链起始信号（ssi）n'-pasH，这对于有效的质粒复制是必不可少的。 cop区域还包含质粒不相容区域inc，包括茎环结构，repA启动子Prep以及两个不同的RepA结合位点BD-1和BD-2。已显示RepA与这些位点的结合方式非常不同，主要以单体或二聚体的形式与BD-1结合以启动RepA转录和质粒复制，而作为BD-2的高级寡聚体以自动调节RepA转录，平衡反映在质粒拷贝中数。活性整合宿主因子（IHF）结合序列位于cop区，并且质粒复制显示依赖于宿主IHF编码基因himA和himD。低浓度的IHF使cop区域易于结合RepA，尽管在反式RepA中高表达时可有效置换结合的IHF，并克服了IHF依赖性。显示不相容性是由于在警察局所在地的RepA滴定引起的，但很容易被过量的RepA所取代。需要两个RepA结合位点来维持不相容性和有效的pKL1复制。没有发现反义RNA和iteron均不参与pKL1调控，因此pKL1是DNA复制自动调控的新例子。当从辅助质粒中过量产生时，RepA诱导pKL1复制到异常高的水平（> 2500拷贝/细胞）。此外，pKL1复制可以被人为调节，并保持广泛的拷贝数。版权所有1999，学术出版社。

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