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Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system.
Gene. 1989 Dec 14; 84(2):209-19.GENE

Abstract

The single-polypeptide RNA polymerases that are encoded by bacteriophage T7 and its relatives form the basis of highly specific and efficient transcription systems. Here, we describe the regulation of transcription from phage promoters by the lac repressor-operator system of Escherichia coli. A synthetic oligodeoxyribonucleotide that contains the core sequence of the lac operator (lacO) was cloned at various distances downstream from the transcription start point (tsp) of the T3 and T7 promoters. The ability of lac repressor to prevent transcription from the phage promoters in vitro was dependent on the position of the operator. Efficient repression was observed when the center of the operator was placed between +14 and +27 (+1 being the tsp), whereas the repressor had little effect when bound to operators centered at +64. For in vivo studies, the chloramphenicol acetyltransferase (CAT)-encoding reporter gene was placed under the control of various promoter-operator constructs, and introduced into bacterial cells containing the genes for the lac repressor and T3 or T7 RNA polymerase. As with in vitro studies, high levels of repression (greater than 4000-fold) of T3 and T7 RNA polymerase activity were achieved, and repression was reversed by the inducer isopropyl-beta-D-thiogalactopyranoside. When the T3 promoter-lacO constructs are used to regulate the expression of a target gene in combination with an inducible RNA polymerase gene under control of the lacUV5 promoter, the doubly regulated system provides extremely tight levels of repression, yet allows high levels of expression after induction. In such a system, we observed a greater than 10(5)-fold increase in CAT activity within 30 min after induction. This system should prove useful in cloning and expressing genes that are potentially toxic to the host cells.

Authors+Show Affiliations

Department of Microbiology and Immunology, Morse Institute for Molecular Biology and Genetics, SUNY-Health Science Center, Brooklyn 11203.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

2693210

Citation

Giordano, T J., et al. "Regulation of Coliphage T3 and T7 RNA Polymerases By the Lac Repressor-operator System." Gene, vol. 84, no. 2, 1989, pp. 209-19.
Giordano TJ, Deuschle U, Bujard H, et al. Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system. Gene. 1989;84(2):209-19.
Giordano, T. J., Deuschle, U., Bujard, H., & McAllister, W. T. (1989). Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system. Gene, 84(2), 209-19.
Giordano TJ, et al. Regulation of Coliphage T3 and T7 RNA Polymerases By the Lac Repressor-operator System. Gene. 1989 Dec 14;84(2):209-19. PubMed PMID: 2693210.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system. AU - Giordano,T J, AU - Deuschle,U, AU - Bujard,H, AU - McAllister,W T, PY - 1989/12/14/pubmed PY - 1989/12/14/medline PY - 1989/12/14/entrez SP - 209 EP - 19 JF - Gene JO - Gene VL - 84 IS - 2 N2 - The single-polypeptide RNA polymerases that are encoded by bacteriophage T7 and its relatives form the basis of highly specific and efficient transcription systems. Here, we describe the regulation of transcription from phage promoters by the lac repressor-operator system of Escherichia coli. A synthetic oligodeoxyribonucleotide that contains the core sequence of the lac operator (lacO) was cloned at various distances downstream from the transcription start point (tsp) of the T3 and T7 promoters. The ability of lac repressor to prevent transcription from the phage promoters in vitro was dependent on the position of the operator. Efficient repression was observed when the center of the operator was placed between +14 and +27 (+1 being the tsp), whereas the repressor had little effect when bound to operators centered at +64. For in vivo studies, the chloramphenicol acetyltransferase (CAT)-encoding reporter gene was placed under the control of various promoter-operator constructs, and introduced into bacterial cells containing the genes for the lac repressor and T3 or T7 RNA polymerase. As with in vitro studies, high levels of repression (greater than 4000-fold) of T3 and T7 RNA polymerase activity were achieved, and repression was reversed by the inducer isopropyl-beta-D-thiogalactopyranoside. When the T3 promoter-lacO constructs are used to regulate the expression of a target gene in combination with an inducible RNA polymerase gene under control of the lacUV5 promoter, the doubly regulated system provides extremely tight levels of repression, yet allows high levels of expression after induction. In such a system, we observed a greater than 10(5)-fold increase in CAT activity within 30 min after induction. This system should prove useful in cloning and expressing genes that are potentially toxic to the host cells. SN - 0378-1119 UR - https://www.unboundmedicine.com/medline/citation/2693210/Regulation_of_coliphage_T3_and_T7_RNA_polymerases_by_the_lac_repressor_operator_system_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0378-1119(89)90494-0 DB - PRIME DP - Unbound Medicine ER -