Praszkier J, Murthy S, Pittard A J
Department of Microbiology and Immunology, The University of Melbourne, Victoria, Australia.
J Bacteriol. 2000 Jul;182(14):3972-80. doi: 10.1128/JB.182.14.3972-3980.2000.
RepA, the replication initiator protein of the IncB plasmid pMU720, acts preferentially in cis. The cis activity of RepA is thought to be mediated by CIS, a 166-bp region of DNA separating the coding region of repA from the origin of replication (ori) of pMU720. To investigate the trans activity of RepA, the repA gene, without its cognate ori, was cloned on a multicopy plasmid, pSU39. The ori on which RepA acts was cloned on pAM34, a plasmid whose replicon is inactive without induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). Thus, in the absence of IPTG, replication of the pAM34 derivatives was dependent on activation of the cloned ori by RepA produced in trans from the pSU39 derivatives. The effect of CIS, when present either on the RepA-producing or the ori plasmid or both, on the efficiency of replication of the ori plasmid in vivo, was determined. The presence of CIS, in its native position and orientation, on the RepA-producing plasmid reduced the efficiency of replication of the ori plasmid. This inhibitory activity of CIS was sequence specific and involved interaction with the C-terminal 20 to 37 amino acids of RepA. By contrast, CIS had no effect when present on the ori plasmid. Initiation of replication from the ori in trans was independent of transcription into CIS.
RepA是IncB质粒pMU720的复制起始蛋白，优先以顺式作用。RepA的顺式活性被认为是由CIS介导的，CIS是一段166bp的DNA区域，将repA的编码区与pMU720的复制起点（ori）分隔开。为了研究RepA的反式活性，将不含其同源ori的repA基因克隆到多拷贝质粒pSU39上。RepA作用的ori被克隆到pAM34上，pAM34是一种质粒，其复制子在没有异丙基-β-D-硫代半乳糖苷（IPTG）诱导时无活性。因此，在没有IPTG的情况下，pAM34衍生物的复制依赖于从pSU39衍生物反式产生的RepA对克隆的ori的激活。测定了CIS在产生RepA的质粒上、ori质粒上或两者上存在时，对ori质粒在体内复制效率的影响。在产生RepA的质粒上，处于其天然位置和方向的CIS的存在降低了ori质粒的复制效率。CIS的这种抑制活性具有序列特异性，并且涉及与RepA的C末端20至37个氨基酸的相互作用。相比之下，CIS存在于ori质粒上时没有影响。从ori反式进行的复制起始与转录进入CIS无关。
