Monomer–dimer control of the ColE1 P cer promoter

Abstract

XerCD-mediated recombination at cer converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. In addition to XerCD, recombination requires the accessory factors ArgR and PepA. The promoter P cer , located centrally within cer, is also required for stable plasmid maintenance. P cer is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division. It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides. This study has shown that ArgR does not act as a transcriptional repressor of P cer in plasmid monomers. P cer is unusual in that the N35 and N10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity. An increase to 17 bp resulted in a 10-to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp. These observations are consistent with the hypothesis that P cer activation involves realignment of the N35 and N10 sequences within a recombinational synaptic complex. This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.

tRNA-dependent cleavage of the ColE1 plasmid-encoded RNA I

“…ColE1-like plasmids use only enzymes encoded by the host, Escherichia coli, for their replication (Cesareni et al, 1991;Sharpe et al, 1999;Chatwin & Summers, 2001;Kim et al, 2005a). This replication is initiated by a transcript, called RNA II, which forms a persistent hybrid with its template DNA and acts as a pre-primer RNA.…”

The role of FIS in the Rcd checkpoint and stable maintenance of plasmid ColE1

“…We suggested previously (Chatwin & Summers, 2001) that P cer is inactive in monomers because of the suboptimal alignment of the 235 and 210 boxes. Promoter activation in dimers was thought to result from the restoration of an optimal alignment by twisting or bending of the promoter in the synaptic complex between two cer sites in a dimer.…”

“…During replication of a plasmid monomer, any proteins assembled at cer will be transiently displaced, and the temporary loss of FIS and XerCD might be expected to cause a brief burst of transcription from P cer . However, the short spacer of the naked promoter ensures that it presents an unattractive substrate for RNA polymerase (Chatwin & Summers, 2001), thus minimizing unwanted production of Rcd.…”

“…Insertion of 2 bp into the spacer (corresponding to a change in the angular separation of the 210 and 235 boxes of approximately 70 u ) results in a 10-to 20-fold increase in transcription. This has led to the proposal that twisting of the promoter in the synaptic complex formed by XerCD, ArgR, PepA and the two cer sites in a plasmid dimer realigns the 235 and 210 regions, and activates Rcd transcription (Chatwin & Summers, 2001).…”

Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac− to Lac+

“…Interestingly, the lacTp promoter sequence of the class III revertants was exactly the same as that of the functional lacTp promoter described for L. casei BL23 . This reflects the fact that maximum promoter activity and open complex formation by RNA polymerase usually happen with promoters in which the length of spacer between the 235 and 210 boxes is 17 bp (Berman & Landy, 1979;Ackerson & Gralla, 1983;Mandecki & Reznikoff, 1982;Stefano & Gralla, 1982;Aoyama et al, 1983;Mandecki et al, 1985;Chatwin & Summers, 2001). For class IV, V and VI revertants, a point mutation was found in the 210 (TATAAC), 210 (TAAACT) and 235 (TTGACA) boxes of the lacTp promoter, respectively.…”