Ouazzani Chahdi A, Gabant P, Couturier M
Laboratoire de Génétique des Procaryotes, Département de Biologie Moléculaire, Université Libre de Bruxelles, Belgium.
Mol Gen Genet. 1997 Aug;255(5):477-86. doi: 10.1007/s004380050520.
RepHI1B is one of the replicons that is specific to IncHI1 multireplicon plasmids. Its general organization resembles that of several replicons that control their copy number by an iteron mechanism. The RepHI1B replicon (2.4 kb) contains: (i) an 882 bp repA gene coding for a 32 kDa replication protein (RepA), sharing significant similarity with the initiator proteins of other replicons belonging to various incompatibility (Inc) groups, including P1 (IncY), Rts1 (IncT), RepFIB (IncFI), and RepHI1A (IncHI1); (ii) two sets of 17 bp DNA repeats (iterons), one upstream and one downstream from repA. By complementation testing, we identified the replication origin (ori) of RepHI1B in a 223 bp locus upstream from repA. By primer extension we mapped two promoters of repA (Pr1 and Pr2) in the ori sequence. We used repA::lacZ transcriptional fusions to study regulation of the repA gene. This analysis showed that repA is transcriptionally autoregulated. Gel mobility shift assays demonstrated that RepA binds specifically to the origin and to iterons overlapping the Pr1 and Pr2 promoters. A G to A transition at nucleotide position 13 of the iteron located in Pr2 (repeat 5) drastically decreases autoregulation of repA by inhibiting binding of RepA.
RepHI1B是IncHI1多复制子质粒特有的复制子之一。其总体结构类似于通过迭代子机制控制其拷贝数的几个复制子。RepHI1B复制子（2.4 kb）包含：（i）一个882 bp的repA基因，编码一个32 kDa的复制蛋白（RepA），与属于不同不相容性（Inc）组的其他复制子的起始蛋白具有显著相似性，包括P1（IncY）、Rts1（IncT）、RepFIB（IncFI）和RepHI1A（IncHI1）；（ii）两组17 bp的DNA重复序列（迭代子），一组在repA上游，一组在repA下游。通过互补测试，我们在repA上游223 bp的位点鉴定出了RepHI1B的复制起点（ori）。通过引物延伸，我们在ori序列中定位了repA的两个启动子（Pr1和Pr2）。我们使用repA::lacZ转录融合来研究repA基因的调控。该分析表明repA受到转录自调控。凝胶迁移率变动分析表明，RepA特异性结合起点以及与Pr1和Pr2启动子重叠的迭代子。位于Pr2（重复序列5）中的迭代子第13位核苷酸处的G到A转换通过抑制RepA的结合，极大地降低了repA的自调控。
