RNAII transcribed by IPTG-induced T7 RNA polymerase is non-functional as a replication primer for ColE1-type plasmids in Escherichia coli.
RNAII, an RNA species encoded by ColE1-type plasmids, serves as a primer for plasmid DNA replication. Previous work has shown that overproduction of RNAII transcribed by Escherichia coli RNA polymerase results in elevated plasmid copy number. To produce a plasmid in which the elevation of its copy number is inducible, we placed transcription of RNAII under the control of a bacteriophage T7 late promoter regulated by IPTG-inducible T7 RNA polymerase. During induction of T7 RNA polymerase by IPTG, we found that RNAII was overexpressed, but that, surprisingly, this increase in RNAII did not result in elevation of plasmid copy number. These results suggest that RNAII transcribed by T7 RNA polymerase does not function as a primer for plasmid DNA replication. Since RNAII function requires folding of its multiple stem-loop structures in a precise conformation and folding of RNAII can be influenced by its rate of transcription, the extremely rapid rate of travel of the T7 RNA polymerase may preclude proper folding of RNAII during its elongation.
Chao
M Y
MY
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Kan
M C
MC
Lin-Chao
S
S
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003087:Colicins / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers; D004261:DNA Replication; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012313:RNA / Q000096:biosynthesis*; D012329:RNA, Bacterial / Q000096:biosynthesis*; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014764:Viral Proteins
D003087:Colicins; D017931:DNA Primers; C034477:RNA primers; D012329:RNA, Bacterial; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases