Abstract
DNA from a transducing bacteriophage carrying a fusion of the tryptophan and lactose operons of E. coli (λdtrp-lac) has been used to direct cell-free synthesis of β-galactosidase (EC 3.2.1.23). Whereas normal lac operon (λdlac) DNA requires adenosine-3′:5′-cyclic monophosphate (cAMP) for β-galactosidase synthesis, trp-lac DNA is unaffected by cAMP. This difference in cAMP dependence verifies the presence of a cAMP-requiring promoter in the lac operon that has been removed from the trp-lac DNA. Synthesis with trp-lac DNA is controlled by the protein product of the tryptophan repressor gene (trpR). Synthesis in extracts of trpR- (repressor-negative) cells is progressively reduced by increased additions of extract from trpR+ cells. No trpR- product repression is seen when β-galactosidase synthesis is programmed by normal lac DNA. This highly sensitive and specific assay has facilitated quantitation and partial purification of the trp repressor.
Keywords: β-galactosidase, transducing bacteriophage, cell-free extract, cyclic AMP, lac operon
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Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- COHEN G., JACOB F. Sur la répression de la synthèse des enzymes intervenant dans la formation du tryptophane chez Escherichia coll. C R Hebd Seances Acad Sci. 1959 Jun 15;248(24):3490–3492. [PubMed] [Google Scholar]
- Doolittle W. F., Yanofsky C. Mutants of Escherichia coli with an altered tryptophanyl-transfer ribonucleic acid synthetase. J Bacteriol. 1968 Apr;95(4):1283–1294. doi: 10.1128/jb.95.4.1283-1294.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eron L., Morse D., Reznikoff W., Beckwith J. Fusions of the lac and trp regions of escherichia coli: covalently fused messenger RNA. J Mol Biol. 1971 Aug 28;60(1):203–209. doi: 10.1016/0022-2836(71)90458-x. [DOI] [PubMed] [Google Scholar]
- Gilbert W., Müller-Hill B. Isolation of the lac repressor. Proc Natl Acad Sci U S A. 1966 Dec;56(6):1891–1898. doi: 10.1073/pnas.56.6.1891. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greenblatt J., Schleif R. Arabinose C protein: regulation of the arabinose operon in vitro. Nat New Biol. 1971 Oct 6;233(40):166–170. doi: 10.1038/newbio233166a0. [DOI] [PubMed] [Google Scholar]
- Hiraga S. Operator mutants of the tryptophan operon in Escherichia coli. J Mol Biol. 1969 Jan 14;39(1):159–179. doi: 10.1016/0022-2836(69)90340-4. [DOI] [PubMed] [Google Scholar]
- Morse D. E., Yanofsky C. Amber mutants of the trpR regulatory gene. J Mol Biol. 1969 Aug 28;44(1):185–193. doi: 10.1016/0022-2836(69)90413-6. [DOI] [PubMed] [Google Scholar]
- Mosteller R. D., Yanofsky C. Evidence that tryptophanyl transfer ribonucleic acid is not the corepressor of the tryptophan operon of Escherichia coli. J Bacteriol. 1971 Jan;105(1):268–275. doi: 10.1128/jb.105.1.268-275.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nisseley S. P., Anderson W. B., Gottesman M. E., Perlman R. L., Pastan I. In vitro transcription of the gal operon requires cyclic adenosine monophosphate and cyclic adenosine monophosphate receptor protein. J Biol Chem. 1971 Aug 10;246(15):4671–4678. [PubMed] [Google Scholar]
- Reznikoff W. S., Miller J. H., Scaife J. G., Beckwith J. R. A mechanism for repressor action. J Mol Biol. 1969 Jul 14;43(1):201–213. doi: 10.1016/0022-2836(69)90089-8. [DOI] [PubMed] [Google Scholar]
- Yanofsky C., Ito J. Nonsense codons and polarity in the tryptophan operon. J Mol Biol. 1966 Nov 14;21(2):313–334. doi: 10.1016/0022-2836(66)90102-1. [DOI] [PubMed] [Google Scholar]
- Zubay G., Gielow L., Englesberg E. Cell-free studies on the regulation of the arabinose operon. Nat New Biol. 1971 Oct 6;233(40):164–165. doi: 10.1038/newbio233164a0. [DOI] [PubMed] [Google Scholar]
- Zubay G., Schwartz D., Beckwith J. Mechanism of activation of catabolite-sensitive genes: a positive control system. Proc Natl Acad Sci U S A. 1970 May;66(1):104–110. doi: 10.1073/pnas.66.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]