T7 RNA polymerase directed expression of the Escherichia coli rrnB operon.
Plasmids having Escherichia coli ribosomal DNA sequences under control of a promoter for T7 RNA polymerase have been constructed. Transcription of the rDNA sequences is dependent on T7 RNA polymerase because the tandem promoters for E. coli RNA polymerase, normally used to direct transcription of these sequences, have been removed. The entire 16S, 23S and 5S coding sequences from the rrnB operon can be efficiently transcribed by T7 RNA polymerase in vitro to yield full-length 30S precursor RNA. When such plasmids are placed into an E. coli strain containing a chromosomal copy of the gene for T7 RNA polymerase under control of the lac UV5 promoter, high-level synthesis of rRNAs from the plasmid can be induced by adding IPTG to exponentially growing cells. Subsequent addition of rifampicin to inhibit further initiation of transcription by E. coli RNA polymerase provides a simple method to study the fate of plasmid-coded rRNAs in the complete absence of host-coded rRNA synthesis. Gel electrophoretic analysis demonstrated that the rRNAs synthesized by T7 RNA polymerase in the presence of rifampicin are processed to their mature forms and assembled into ribosomal particles for at least 35 min after rifampicin addition. T7 RNA polymerase is also capable of efficient transcription of the entire rrnB operon in the reverse direction.
D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004275:DNA, Ribosomal / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012335:RNA, Ribosomal / Q000096:biosynthesis; D013604:T-Phages / Q000201:enzymology*
D004274:DNA, Recombinant; D004275:DNA, Ribosomal; D012335:RNA, Ribosomal; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes