Viability of E. coli cells containing phage RNA polymerase and promoter: interference of plasmid replication by transcription.
Strong transcription of phage promoters often renders the host E. coli cells containing the phage RNA polymerase inviable. When expression of the phage SP6 RNA polymerase gene in one plasmid was induced in the E. coli JM109 cells, cells that bear an active SP6 promoter were inviable. When it was not induced (the polymerase was still produced in low levels), viability of the host cells and stability of the promoter-bearing plasmids depended on the orientation of the promoter with respect to that of the replication origin and on the sequence of the origin. A group of SP6 promoter-bearing plasmids (group I plasmids) that had the promoter directed towards the ColE1 replication origin, rendered the polymerase-containing host cells inviable in selective media. When the sequence of the origin was different (group II plasmids), this adverse effect was not observed. When the promoter direction was same as the replication origin and the ampicillin-resistant gene (group III plasmids), many satellites formed around the colonies on ampicillin-containing agar plates. These effects were caused by strong transcription of the phage SP6 promoter by its RNA polymerase, since they were reduced or eliminated by inserting an active terminator just downstream of the promoter. The viability of host cells and copy number of the promoter/terminator-bearing plasmids appear to be quantitatively related with efficiency of initiation and termination of the phage transcription. These systems may be useful for in vivo screening for mutant variants of the phage promoter, polymerase and terminator that are affected in their efficiency.
Kwon
Y S
YS
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
Kim
J
J
Kang
C
C
D001435:Bacteriophages / Q000201:enzymology; D004261:DNA Replication; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000166:cytology; D018628:Gene Dosage; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
C070852:RNA polymerase SP6; D012321:DNA-Directed RNA Polymerases