lac repressor-operator interaction

Riggs, Arthur D.; Suzuki, Hiromi; Bourgeois, Suzanne

doi:10.1016/0022-2836(70)90219-6

Cited by 785 publications

(365 citation statements)

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“…Such DNA sequence-specific binding was assessed by nitrocellulose filter-binding assays (27) in which nick-translated placental DNAs which were hemimethylated and m5C-enriched (50% of C residues methylated) or m5C-poor (2% of C residues methylated) were compared as ligands. As seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

1984

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We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally5 has virtually all of its cytosine residues replaced by 5-methylcytosine (m C). Micrococcus luteus DNA wgs just as good a substrate if it was first similarly nick translated with m dCTP instead of dCTP in the polymerization mixture. At different stages in purification and under various conditions (including in the _presence or absence of high mobility group proteins), the methylation of m C-deficient DVA and that of hemimethylated DNA were compared. Although hemimethylated, m C-rich DNAs were much better substrates than were m C-deficient DNAs and normal XP12 DNA could not be methylated, all of these DNAs were bound equally well by the enzyme. In contrast, from the same placental extraSt, a DNAbinding protein of unknown functon was isolated which binds to m C-rich DNA in preference to the analogous m C-poor DNA.

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Section: Resultsmentioning

confidence: 99%

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

1984

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“…The assay measures the retention of 32P-labeled DNA to nitrocellulose filters (B-6, Schleicher and Schuell). The assay mixture contains an excess of unlabeled "chicken blood" DNA (Calbiochem), which competes with the labeled phage DNA for proteins with nonspecific DNA-binding activity (30 was for 30 min at 300. The culture was then chilled, and the cells were collected by centrifugation, resuspended in 11 ml of 50 mM Tris-HCl, pH 7.2/10% sucrose, and quickly frozen in a dry ice/alcohol bath.…”

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confidence: 99%

Characterization of the integration protein of bacteriophage lambda as a site-specific DNA-binding protein.

Kotewicz¹,

Chung²,

Takeda³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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The Int protein specified by bacteriophage X is required for the recombination event (Fig. 1). Excisive recombination differs from integrative recombination in its requirement for the product of the X int and xis genes (9-11). Although these general features of site-specific recombination have been known for some time from genetic experiments, the absence of a biochemical analysis has impeded a further understanding of the basis for specificity and the mechanism regulating the direction of the reaction.The Int protein has been identified radiochemically by acrylamide gel electrophoresis of labeled proteins under denaturing conditions (12, 13). More recently, assays for the complete site-specific recombination reaction in vitro have been described (14)(15)(16) (9,22), xisl and xls6 (9), Sam7 (23). The phage deletion and/or substitution mutations used were: b538, deleting the phage attachment site (24); b522, deleting the int and xis genes but not the phage recombination genes (24); gal8, carrying the bacterial gal operon and the left prophage attachment site ba' (25); blo7-20, carrying the bacterial blo operon and the right prophage attachment site ab' (26); gal8bio7-20, carrying both the gal and bNo genes and the bacterial attachment site bb' (1, 2, 10 and Fig. 1).Preparation of Phage [32P]DNA. 32P-labeled phage DNAs were prepared as described previously (27,28). In brief, cells were grown in 100 ml of low-phosphate medium to a density of 5 X 108 cells per ml, concentrated by centrifugation, and infected with phage carrying the appropriate attachment site and the lysis-defective mutation Sam7 at a multiplicity of 5 phage/cell. After a 15 min adsorption period, the infected cells were poured into fresh low-phosphate medium containing 5 ACi/ml of 32P-labeled inorganic phosphate. The cells were shaken at 370 for 3 hr and then collected by centrifugation and resuspended in 10 ml of 10 mM Tris-HCI, pH 7.2/10 mM MgCl2. The cells were lysed with chloroform, the debris was removed by centrifugation, and the supernatant fraction containing the phage was centrifuged to equilibrium in a CsCl density gradient. The phage band was removed with a syringe and the phage were again centrifuged to equilibrium in CsCl. The phage DNA was extracted by four treatments with redistilled phenol, and the phenol was removed with ether. The DNA was then dialyzed extensively into 10 mM Tris-HCl, pH 7.2/0.1 mM EDTA.DNA-Binding Assay. The details of the DNA-binding assay used for Int protein have been described previously (27)(28)(29). The assay measures the retention of 32P-labeled DNA to nitrocellulose filters (B-6, Schleicher and Schuell). The assay mixture contains an excess of unlabeled "chicken blood" DNA (Calbiochem), which competes with the labeled phage DNA for proteins with nonspecific DNA-binding activity (30

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“…The different intermediary transcription complexes have been postulated from experiments using the radioactive incorporation test [7], kinetic experiments using the filter-binding method [8,9] and the abortive initiation assay [lo]. This paper characterizes some of the intermediary transcription complexes on the basis of their electrophoretic mobility on non-denaturing gels.…”

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Visualization of intermediary transcription states in the complex between Escherichia coli DNA‐dependent RNA polymerases and a promoter‐carrying DNA fragment using the gel retardation method

Heumann

Metzger

Niehörster

1986

European Journal of Biochemistry

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DNA-dependent RNA polymerase in complex with a DNA fragment was analyzed by electrophoresis in nondenaturing gels as core enzyme, holoenzyme, during initiation and elongation. The DNA fragment carried the promoter A1 of the phage T7. The stoichiometry between holoenzyme and promoter and between CJ and core enzyme in complex with DNA was determined. Holoenzyme bound as a monomer to the DNA, whereas core enzyme formed aggregates before binding to the DNA. If the molar ratio of holoenzyme to DNA exceeded 0.5: 1 a second holoenzyme molecule interacted with the DNA fragment with diminished affinity. A large difference in the frictional coefficient of the holoenzyme-promoter and the core enzyme-DNA complex indicated a drastic conformational difference between the two types of complexes. The stability of the holoenzyme-promoter complex decreased with decreasing temperature, accompanied by at least partial dissociation of holoenzyme into core enzyme and CJ factor. Addition of nucleoside triphosphates did not change the electrophoretic mobility of the complex if abortive transcription only was allowed, but increased it after addition of all four nucleoside triphosphates owing to release of the CJ factor.The eubacterial DNA-dependent RNA polymerase (subunit composition: Pj?aza) in complex with a promoter adopts a number of sequential conformational states until an active transcription complex is formed. Four different transient complexes have been proposed to describe the transition from a non-specific RNA-polymerase DNA complex to a specific initiation complex [I -31: (a) a non-specific RNA-polymerase-DNA complex; (b) a specific closed complex; (c) an isomerisation complex as a result of a conformational change of the RNA polymerase; (d) a second isomerisation complex (open complex) as a result of the melting of the DNA.Adding only the starting nucleoside triphosphates, an initiation complex is formed, which catalyzes the synthesis of RNA molecules abortively to a length of approximately 8 nucleotides. After addition of more triphosphates and thus formation of longer transcripts, the initiation factor c is released from the ternary complex [4 -61.The different intermediary transcription complexes have been postulated from experiments using the radioactive incorporation test [7], kinetic experiments using the filter-binding method [8,9] and the abortive initiation assay [lo]. This paper characterizes some of the intermediary transcription complexes on the basis of their electrophoretic mobility on non-denaturing gels. Previously non-denaturing gels were used for the study of the interaction of complexes with long DNA fragments could not be characterized because the protein-DNA complexes did not enter the gel matrix. Smaller DNA fragments were used by Fried and Crothers [12] to discriminate different occupational states of the repressor/operator system. We applied this gel method on the investigation of the interaction of DNA-dependent RNA polymerase with a promoter-carrying DNA fragment. MATERIALS AND METHODSRNA polymera...

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lac repressor-operator interaction

Cited by 785 publications

References 19 publications

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

Characterization of the integration protein of bacteriophage lambda as a site-specific DNA-binding protein.

Visualization of intermediary transcription states in the complex between Escherichia coli DNA‐dependent RNA polymerases and a promoter‐carrying DNA fragment using the gel retardation method

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