Nucleic Acids Research, 1981, Vol. 9, No. 13 3047-3060
© 1981
MOLECULAR BIOLOGY |
A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system+
Department of Biochemistry, Michigan State University, East Lansing MI 48824, USA
Received April 21, 1981.
The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified.
This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promoter, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.
+A preliminary report of this work was presented at the 2nd "Conversation in the Discipline Biomolecular Stereodynamics", Albany, NY, April, 1981.
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L. L. L. Paxton, L.-J. Li, V. Secor, J. L. Duff, S. M. Naik, N. Shibagaki, and S. W. Caughman Flanking Sequences for the Human Intercellular Adhesion Molecule-1 NF-kappa B Response Element Are Necessary for Tumor Necrosis Factor alpha -Induced Gene Expression J. Biol. Chem., June 20, 1997; 272(25): 15928 - 15935. [Abstract] [Full Text] [PDF]
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K. C. Das and C. W. White Activation of NF-kappa B by Antineoplastic Agents. ROLE OF PROTEIN KINASE C J. Biol. Chem., June 6, 1997; 272(23): 14914 - 14920. [Abstract] [Full Text] [PDF]
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D. L. Carlone and J. S. Richards Functional Interactions, Phosphorylation, and Levels of 3',5'-Cyclic Adenosine Monophosphate-Regulatory Element Binding Protein and Steroidogenic Factor-1 Mediate Hormone-Regulated and Constitutive Expression of Aromatase in Gonadal Cells Mol. Endocrinol., March 1, 1997; 11(3): 292 - 304. [Abstract] [Full Text]
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J. Chaudhary, A. S. Cupp, and M. K. Skinner Role of Basic-Helix-Loop-Helix Transcription Factors in Sertoli Cell Differentiation: Identification of an E-Box Response Element in the Transferrin Promoter Endocrinology, February 1, 1997; 138(2): 667 - 675. [Abstract] [Full Text] [PDF]
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T. Grotzinger, K. Jensen, and H. Will The Interferon (IFN)-stimulated Gene Sp100 Promoter Contains an IFN-gamma Activation Site and an Imperfect IFN-stimulated Response Element Which Mediate Type I IFN Inducibility J. Biol. Chem., October 11, 1996; 271(41): 25253 - 25260. [Abstract] [Full Text] [PDF]
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C. R. Alonso, C. G. Pesce, and A. R. Kornblihtt The CCAAT-binding Proteins CP1 and NF-I Cooperate with ATF-2 in the Transcription of the Fibronectin Gene J. Biol. Chem., September 6, 1996; 271(36): 22271 - 22279. [Abstract] [Full Text] [PDF]
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M. Yin, S. Q. Yang, H. Z. Lin, M. D. Lane, S. Chatterjee, and A. M. Diehl Tumor Necrosis Factor alpha Promotes Nuclear Localization of Cytokine-inducible CCAAT/Enhancer Binding Protein Isoforms in Hepatocytes J. Biol. Chem., July 26, 1996; 271(30): 17974 - 17978. [Abstract] [Full Text] [PDF]
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J. K. Morris and J. S. Richards An E-box Region within the Prostaglandin Endoperoxide Synthase-2 (PGS-2) Promoter Is Required for Transcription in Rat Ovarian Granulosa Cells J. Biol. Chem., July 12, 1996; 271(28): 16633 - 16643. [Abstract] [Full Text] [PDF]
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C. L. Parks and T. Shenk The Serotonin 1a Receptor Gene Contains a TATA-less Promoter that Responds to MAZ and Sp1 J. Biol. Chem., February 23, 1996; 271(8): 4417 - 4430. [Abstract] [Full Text] [PDF]
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D. Y. Huang and M. B. Prystowsky Identification of an Essential Cis-Element Near the Transcription Start Site for Transcriptional Activation of the Proliferating Cell Nuclear Antigen Gene J. Biol. Chem., January 12, 1996; 271(2): 1218 - 1225. [Abstract] [Full Text] [PDF]
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J. A. Ripperger, S. Fritz, K. Richter, G. M. Hocke, F. Lottspeich, and G. H. Fey Transcription Factors Stat3 and Stat5b Are Present in Rat Liver Nuclei Late in an Acute Phase Response and Bind Interleukin-6 Response Elements J. Biol. Chem., December 15, 1995; 270(50): 29998 - 30006. [Abstract] [Full Text] [PDF]
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R. C. Muise-Helmericks and N. Rosen Identification of a Novel Repressive Element in the Proximal lck Promoter J. Biol. Chem., November 17, 1995; 270(46): 27538 - 27543. [Abstract] [Full Text] [PDF]
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R.-T. Zong and R. H. Scheuermann Mutually Exclusive Interaction of a Novel Matrix Attachment Region Binding Protein and the NF-µNR Enhancer Repressor J. Biol. Chem., October 13, 1995; 270(41): 24010 - 24018. [Abstract] [Full Text] [PDF]
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M. Thomas, H. Skala, A. Kahn, and F.ço. P. D. Tuy Functional Dissection of the Brain-specific Rat Aldolase C Gene Promoter in Transgenic Mice J. Biol. Chem., September 1, 1995; 270(35): 20316 - 20321. [Abstract] [Full Text] [PDF]
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N. Naidoo, B. S. Cooperman, Z.-m. Wang, X.-z. Liu, and H. Rubin Identification of Lysines within [IMAGE]1-Antichymotrypsin Important for DNA Binding J. Biol. Chem., June 16, 1995; 270(24): 14548 - 14555. [Abstract] [Full Text] [PDF]
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E. A. Eklund and D. G. Skalnik Characterization of a gp91-phox Promoter Element That Is Required for Interferon [IMAGE]-induced Transcription J. Biol. Chem., April 7, 1995; 270(14): 8267 - 8273. [Abstract] [Full Text] [PDF]
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J. Darnell Jr, I. Kerr, and G. Stark Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins Science, June 3, 1994; 264(5164): 1415 - 1421. [Abstract] [PDF]
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D. Paolella, C. Palmer, and A Schepartz DNA targets for certain bZIP proteins distinguished by an intrinsic bend Science, May 20, 1994; 264(5162): 1130 - 1133. [Abstract] [PDF]
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B Cuenoud and A Schepartz Altered specificity of DNA-binding proteins with transition metal dimerization domains Science, January 22, 1993; 259(5094): 510 - 513. [Abstract] [PDF]
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