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T7 RNA polymerase directed expression of the Escherichia coli rrnB operon.
EMBO J 1986; 5(5):1099-103EJ

Abstract

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.

Authors

No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

3013618

Citation

Steen, R, et al. "T7 RNA Polymerase Directed Expression of the Escherichia Coli rrnB Operon." The EMBO Journal, vol. 5, no. 5, 1986, pp. 1099-103.
Steen R, Dahlberg AE, Lade BN, et al. T7 RNA polymerase directed expression of the Escherichia coli rrnB operon. EMBO J. 1986;5(5):1099-103.
Steen, R., Dahlberg, A. E., Lade, B. N., Studier, F. W., & Dunn, J. J. (1986). T7 RNA polymerase directed expression of the Escherichia coli rrnB operon. The EMBO Journal, 5(5), pp. 1099-103.
Steen R, et al. T7 RNA Polymerase Directed Expression of the Escherichia Coli rrnB Operon. EMBO J. 1986;5(5):1099-103. PubMed PMID: 3013618.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - T7 RNA polymerase directed expression of the Escherichia coli rrnB operon. AU - Steen,R, AU - Dahlberg,A E, AU - Lade,B N, AU - Studier,F W, AU - Dunn,J J, PY - 1986/5/1/pubmed PY - 1986/5/1/medline PY - 1986/5/1/entrez SP - 1099 EP - 103 JF - The EMBO journal JO - EMBO J. VL - 5 IS - 5 N2 - 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. SN - 0261-4189 UR - https://www.unboundmedicine.com/medline/citation/3013618/T7_RNA_polymerase_directed_expression_of_the_Escherichia_coli_rrnB_operon_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/3013618/ DB - PRIME DP - Unbound Medicine ER -