Womble D D, Dong X, Wu R P, Luckow V A, Martinez A F, Rownd R H
J Bacteriol. 1984 Oct;160(1):28-35. doi: 10.1128/jb.160.1.28-35.1984.
The region of DNA coding for incompatibility (inc) and copy number control (cop) of the IncFII plasmid NR1 is transcribed in both the rightward and leftward directions. The rightward transcripts serve as mRNA for the repA1 protein, which is required for replication. A small, 91-base leftward transcript is synthesized from the opposite DNA strand and is complementary to a portion of the rightward mRNA near its 5' end. A 262-base-pair Sau3A restriction fragment that encodes the small leftward transcript, but does not include the rightward transcription promoters, was cloned into the vector pBR322 or pUC8. The same fragment was cloned from an Inc- mutant of NR1 that does not make the small leftward transcript. Transcription through the cloned fragments in these derivatives was under control of the tetracycline resistance gene in pBR322 or the lac promoter-operator in pUC8. In one orientation of the inserted DNA, a hybrid transcript containing rightward NR1 RNA sequences was synthesized. In the other orientation, a hybrid transcript containing leftward NR1 RNA sequences was synthesized. These plasmids were used to vary the intracellular levels of the rightward or leftward NR1 RNA transcripts and to test their effects in trans on various coresident derivatives of NR1. An excess of rightward NR1 RNA in trans stimulated expression of the essential repA1 gene and caused an increase in the copy number of a coresident NR1 plasmid. An excess of leftward NR1 RNA in trans inhibited the expression of the repA1 gene and lowered the coresident NR1 copy number, thereby causing incompatibility. A pBR322 derivative with no transcription through the cloned NR1 DNA had no effect in trans. These results suggest that the small leftward transcript is the incompatibility inhibitor of NR1 and that its target is the complementary portion of the rightward mRNA.
IncFII质粒NR1的不相容性（inc）和拷贝数控制（cop）的DNA编码区域可向左右两个方向转录。向右的转录本作为repA1蛋白的mRNA，该蛋白是复制所必需的。一个由91个碱基组成的小向左转录本从相反的DNA链合成，并且与向右mRNA 5'端附近的一部分互补。一个编码小向左转录本但不包括向右转录启动子的262个碱基对的Sau3A限制性片段被克隆到载体pBR322或pUC8中。从不产生小向左转录本的NR1的Inc-突变体中克隆出相同的片段。这些衍生物中通过克隆片段的转录受pBR322中的四环素抗性基因或pUC8中的lac启动子-操纵子控制。在插入DNA的一个方向上，合成了包含向右NR1 RNA序列的杂合转录本。在另一个方向上，合成了包含向左NR1 RNA序列的杂合转录本。这些质粒用于改变向右或向左NR1 RNA转录本的细胞内水平，并测试它们对NR1各种共居衍生物的反式作用。过量的向右NR1 RNA反式刺激必需的repA1基因的表达，并导致共居的NR1质粒拷贝数增加。过量的向左NR1 RNA反式抑制repA1基因的表达并降低共居的NR1拷贝数，从而导致不相容性。没有通过克隆的NR1 DNA转录的pBR322衍生物反式没有作用。这些结果表明，小向左转录本是NR1的不相容性抑制剂，其靶标是向右mRNA的互补部分。
