The majority of large multiresistance plasmids of Staphylococcus aureus utilise a RepA_N-type replication initiation protein, the expression of which is regulated by a small antisense RNA (RNAI) that overlaps the rep mRNA leader. The pSK41/pGO1-family of conjugative plasmids additionally possess a small (86 codon) divergently transcribed ORF (orf86) located upstream of the rep locus. The product of pSK41 orf86 was predicted to have a helix-turn-helix motif suggestive of a likely function in transcriptional repression. In this study, we investigated the effect of Orf86 on transcription of thirteen pSK41 backbone promoters. We found that Orf86 only repressed transcription from the rep promoter, and hence now redesignate the product as Cop. Over-expression of Cop in trans reduced the copy number of pSK41 mini-replicons, both in the presence and absence of rnaI. in vitro protein-DNA binding experiments with purified 6 × His-Cop demonstrated specific DNA binding, adjacent to, and partially overlapping the −35 hexamer of the rep promoter. The crystal structure of Cop revealed a dimeric structure similar to other known transcriptional regulators. Cop mRNA was found to result from “read-through” transcription from the strong RNAI promoter that escapes the rnaI terminator. Thus, P_rnaI is responsible for transcription of two distinct negative regulators of plasmid copy number; the antisense RNAI that primarily represses Rep translation, and Cop protein that can repress rep transcription. Deletion of cop in a native plasmid did not appear to impact copy number, indicating a cryptic auxiliary role.

大多数金黄色葡萄球菌的大型多抗性质粒都利用 RepA_N 型复制起始蛋白，其表达受到与rep mRNA 前导重叠的小反义 RNA (RNAI) 的调节。pSK41/pGO1 接合质粒家族另外还具有位于rep基因座上游的小（86 个密码子）不同转录的 ORF ( orf86 ) 。pSK41 orf86的产物预计具有螺旋-转角-螺旋基序，暗示其可能在转录抑制中发挥作用。在本研究中，我们研究了 Orf86 对 13 个 pSK41 主链启动子转录的影响。我们发现Orf86仅抑制rep启动子的转录，因此现在将该产物重新命名为Cop。无论存在还是不存在rnaI ， Cop反式过度表达都会减少 pSK41 微型复制子的拷贝数。使用纯化的 6 × His-Cop 进行的体外蛋白质-DNA 结合实验表明，与rep启动子的-35六聚体相邻且部分重叠的特异性DNA结合。Cop 的晶体结构揭示了与其他已知转录调节因子类似的二聚体结构。人们发现Cop mRNA 是由强 RNAI 启动子“通读”转录产生的，该启动子逃脱了RNAI终止子。因此，P _rnaI负责转录两个不同的质粒拷贝数负调节因子；主要抑制Rep翻译的反义RNAI和可以抑制rep转录的Cop蛋白。天然质粒中cop的删除似乎不会影响拷贝数，表明其具有神秘的辅助作用。