IncFII plasmid incompatibility product and its target are both RNA transcripts

Abstract

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.