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 NRl is transcribed in both the rightward and leftward directions. The rightward transcripts serve as mRNA for the repAl 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-basepair 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 Incmutant of NRl 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 NRl RNA sequences was synthesized. In the other orientation, a hybrid transcript containing leftward NRl RNA sequences was synthesized. These plasmids were used to vary the intracellular levels of the rightward or leftward NRl RNA transcripts and to test their effects in trans on various coresident derivatives of NRl. An excess of rightward NR1 RNA in trans stimulated expression of the essential repAl gene and caused an increase in the copy number of a coresident NR1 plasmid. An excess of leftward NRl RNA in trans inhibited the expression of the repAl gene and lowered the coresident NR1 copy number, thereby causing incompatibility. A pBR322 derivative with no transcription through the cloned NRl DNA had no effect in trans. These results suggest that the small leftward transcript is the incompatibility inhibitor of NRl and that its target is the complementary portion of the rightward mRNA.

Control of replication of plasmid R1: translation of the 7k reading frame in the RepA mRNA leader region counteracts the interaction between CopA RNA and CopT RNA.

“…To avoid this we used the plasmid pKN417 which contains the complete 7k gene and its promoter, but also carries a mutant CopA promoter which results in a more than 50-fold decrease in CopA RNA production . The expected effect of RepA mRNA production from such a plasmid, irrespective of putative 7k protein activity, is a titration of CopA RNA produced from pGW98 (see Light and Molin, 1983;Womble et al, 1984), which would in turn stimulate RepA-LacZ translation. Therefore, to detect any possible effect of the 7k protein, we had to correct for the titration of CopA RNA by the additional target sequences.…”

Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III.

“…Its upstream segment encodes the transcriptional repressor CopB, which shuts off the repA promoter almost completely. The main control system that is used to measure the concentration of the plasmid and to adjust the replication frequency accordingly, is acting post-transcriptionally (Light and Molin, 1983;Womble et al, 1984). Its key element is a small, unstable, untranslated RNA (CopA) which forms an RNA duplex with its complementary region in the RepA mRNA; the target for CopA binding (CopT) is upstream of the RepA coding sequence.…”

Establishment of uncharacterized plasmids in Escherichia coli by in vitro transposition

“…3) when 1 mM IPTG was added to an exponentially growing culture. A second plasmid carrying lacI q [13], pVLRR10 [14], ensured control of the inducible replicon. This plasmid also serves as an internal loading control, showing that plasmid DNA from an equal number of cells was run in each lane.…”