Flexibility in repression and cooperativity by KorB of broad host range IncP-1 plasmid RK2
Flexibility in repression and cooperativity by KorB of broad host range IncP-1 plasmid RK2
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DOI：
10.1016/j.jmb.2005.03.062
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发表时间：
2005-06-03
影响因子：
5.6
通讯作者：
Thomas, CM
中科院分区：
文献类型：
--
作者：
Bingle, LEH;Macartney, DP;Thomas, CM
由质粒RK2编码的KorB属于活性分配蛋白的ParB家族。它与RK2基因组上的12个操纵子结合，先前已知它能抑制紧邻操纵子O(B)1、O(B)10和O(B)12（近端）的启动子，或者抑制距离O(B)2、O(B)9和O(B)11达154bp远（远端）的启动子。为了实现强抑制，KorB需要与另外两种由质粒编码的阻遏物KorA或TrbA中的一种进行协同相互作用。本研究使用报告基因检测来测试额外的KorB操纵子是否会影响转录以及测试KorB如何远距离作用。O(B)9和trbBp之间的距离可以增加到1.6kb，而抑制作用或与TrbA的协同性几乎没有降低。当OB位点置于trbBp下游时，KorB也能够抑制启动子并与TrbA协同作用。这表明位于距离RK2上任何已知启动子很远的OB位点具有潜在的调控作用。位于traGp上游1.9kb的O(B)4被证明可介导TrbA增强的KorB对该启动子的抑制，但未观察到对O(B)4上游的traJp有影响，这可能是由于在相邻的转移起始位点oriT形成的核蛋白复合物的阻碍或拓扑学影响。当在LacI(+)宿主的情况下在O(B)9和trbBp之间插入一个lac操纵子时，抑制作用和协同性显著减弱，这是检测DNA结合蛋白扩散的标准测试。另一方面，DNA成环的标准测试，即操纵子移动到与天然结合位点相对的DNA螺旋面，在相对较短的距离内并没有显著影响KorB的抑制作用或与TrbA和KorA的协同性。虽然这些结果与扩散作为KorB到达其靶标的机制更相符，但先前对每个细胞中KorB分子的估计与从每个OB扩散多达1kb所需的数量不一致。因此，一个合理的模型是KorB两者都能做到，即在相对较短的距离内扩散以及在较长距离内成环。（c）2005 Elsevier Ltd.保留所有权利。
KorB, encoded by plasmid RK2, belongs to the ParB family of active partitioning proteins. It binds to 12 operators on the RK2 genome and was previously known to repress promoters immediately adjacent to operators O(B)1, O(B)10 and O(B)12 (proximal) or up to 154 bp away (distal) from O(B)2, O(B)9 and O(B)11. To achieve strong repression, KorB requires a cooperative interaction with one of two other plasmid-encoded repressors, KorA or TrbA. Reporter gene assays were used in this study to test whether the additional KorB operators may influence transcription and to test how KorB acts at a distance. The distance between O(B)9 and trbBp could be increased to 1.6 kb with little reduction in repression or cooperativity with TrbA. KorB was also able to repress the promoter and cooperate with TrbA when the OB site was placed downstream of trbBp. This suggested a potential regulatory role for OB sites located a long way from any known promoter on RK2. O(B)4, 1.9 kb upstream of traGp, was shown to mediate TrbA-potentiated KorB repression of this promoter, but no effect on traJp upstream of O(B)4 was observed, which may be due to the roadblocking or topological influence of the nucleoprotein complex formed at the adjacent transfer origin, oriT. Repression and cooperativity were alleviated significantly when a lac operator was inserted between O(B)9 and trbBp in the context of a LacI(+) host, a standard test for spreading of a DNA-binding protein. On the other hand, a standard test for DNA looping, movement of the operator to the opposite face of the DNA helix from the natural binding site, did not significantly affect KorB repression or cooperativity with TrbA and KorA over relatively short distances. While these results are more consistent with spreading as the mechanism by which KorB reaches its target, previous estimates of KorB molecules per cell are not consistent with there being enough to spread up to 1 kb from each OB. A plausible model is therefore that KorB can do both, spreading over relatively short distances and looping over longer distances. (c) 2005 Elsevier Ltd. All rights reserved.