Online citations, reference lists, and bibliographies.
Referencing for people who value simplicity, privacy, and speed.
Get Citationsy
← Back to Search

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

M. Garner, A. Revzin
Published 1981 · Biology, Medicine, Computer Science

Save to my Library
Download PDF
Analyze on Scholarcy Visualize in Litmaps
Reduce the time it takes to create your bibliography by a factor of 10 by using the world’s favourite reference manager
Time to take this seriously.
Get Citationsy
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 promotor, 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.
This paper references
RNA polymerase
W. Mangel (1977)
The nucleotide sequence of the lactose messenger ribonucleic acid transcribed from the UV5 promoter mutant of Escherichia coli.
N. Maizels (1973)
New Controlling Element in the Lac Operon of E. coli
K. Ippen (1968)
J . Mol. B i o l
D C Hinkle (1972)
Proc. Natl. Acad. Sci. USA 66
A E Silverstone (1970)
Preparation and characterization of large amounts of restriction fragments containing the E. coli lac control elements.
S. Hardies (1979)
D E Johnston (1976)
Biochemistry Nucl. Acids Res
S A Saxe (1979)
Biochim. Biophys. Acta
T Boone (1978)
A rd i t t i
A. E. Silverstone (1970)
Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol.
D. Clewell (1972)
Purification and DNA-binding properties of the catabolite gene activator protein.
A. Riggs (1971)
Proc. Natl. Acad. Sci. USA Proc. Natl. Acad. Sci. USA 72
C L Cech (1973)
J . Mol. B i o l
W Berg (1966)
Acad. S c i
Hawley (1980)
Catabolite-insensitive revertants of lac promoter mutants.
A. Silverstone (1970)
Biochim. Biophys. Acta
D D Anthony (1970)
Bact. Revs
I Pastan (1976)
Eur. J . Biochem
G Walter (1967)
A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography.
R. Burgess (1975)
Rate-limiting steps in RNA chain initiation.
W. McClure (1980)
Proc. N a t l . Acad. S c i . USA 68
A D Riggs (1971)
J . Biol. Chem
W B Anderson (1971)
Studies with the RNA polymerase. 3. Enzymatic activity of the monomer form.
D. Anthony (1970)
Initiation of in vitro mRNA synthesis from the wild-type lac promoter.
J. Majors (1975)
In vitro comparison of initiation properties of bacteriophage lambda wild-type PR and x3 mutant promoters.
D. Hawley (1980)
A neighbor-interaction analysis of the hypochromism and spectra of DNA.
G. Felsenfeld (1965)
Acad. S c i
W R Mcclure (1980)
J . Mol. B i o l
A D Riggs (1970)
J . Mol. B i o l
A D Riggs (1968)
Specific binding of CAP factor to lac promoter DNA
J. Majors (1975)
A quantitative assay for bacterial RNA polymerases.
M. Chamberlin (1979)
J . B i o l . Chem
M J Chamberlin (1978)
Biochemistry Biochemistry
R R Burgess (1975)
A rapid high-yield purification procedure for the cyclic adenosine 3',5'-monophosphate receptor protein from Escherichia coli.
T. Boone (1978)
Characterization of ribonucleic acid polymerase-T7 promoter binary complexes.
C. L. Cech (1980)
F. G. Young (1955)
Cooperative binding to DNA of catabolite activator protein of Escherichia coli.
S. Saxe (1979)
J. Mol. Biol. Eur. J. Biochem
L E Maquat (1977)
J . Mol. B i o l
D C H I N K L E (1970)
Quantitation of the interaction of EScherichia coli RNA polymerase holoenzyme with double-helical DNA using a thermodynamically rigorous centrifugation method.
A. Revzin (1981)
Non-specific interactions of CRP from E. coli with native and denatured DNAs: control of binding by cAMP and cGMP and by cation concentration.
M. Takahashi (1979)
Ann.Rev. Biochem. Nucl. Acids Res
M J Chamberlin (1974)
DNA binding of the lac repressor.
A. Riggs (1968)
Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase.
P. Lowe (1979)
Studies on the binding of RNA polymerase to polynucleotides.
O. Jones (1966)
Studies of the binding of Escherichia coli RNA polymerase to DNA. I. The role of sigma subunit in site selection.
D. Hinkle (1972)
Studies of the binding of Escherichia coli RNA polymerase to DNA. II. The kinetics of the binding reaction.
D. Hinkle (1972)
Initiation of DNA-dependent RNA synthesis and the effect of heparin on RNA polymerase.
G. Walter (1967)
In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters.
L. Maquat (1978)
The lac repressor-operator interaction. 3. Kinetic studies.
A. Riggs (1970)
Cyclic adenosine 5'-monophosphate in Escherichia coli.
I. Pastan (1976)
Lac repressor-operator interaction. I. Equilibrium studies.
A. Riggs (1970)
Interaction of RNA polymerase with promoters from bacteriophage fd.
P. Seeburg (1977)

This paper is referenced by
Structure of the isocitrate lyase gene of cucumber and its transcriptional regulation in higher plants
S. J. Reynolds (1994)
Computational Methods for the Measurement of Protein-DNA Interactions
D. James (2018)
Mechanisms by which variants in the TCF7L2 gene increase the risk of developing Type 2 diabetes
Yuedan Zhou (2013)
Electrophoretic Mobility Shift Assay (EMSA).
M. Smith (2001)
proteins using a simple proteomics approach that
J. A. Stead (2006)
Highly sensitive SERS quantification of the oncogenic protein c-Jun in cellular extracts.
L. Guerrini (2013)
Advances in the Discovery of cis-Regulatory Elements
Y. Pan (2006)
Sequences required for in vitro transcriptional activation of a Drosophila hsp 70 gene
J. Topol (1985)
Detection of 12-o-tetradecanoylphorbol-13-acetate-induced cellular proteins that compete with the Epstein-Barr virus nuclear antigen 1 (EBNA-1) for binding to a site within the Epstein-Barr virus oriP.
M. Nonoyama (1990)
The gene blistered selectively controls muscle type differentiation in Drosophila melanogaster
Ashley A DeAguero (2015)
Protein–Oligonucleotide Interactions
O. Gabrielsen (2006)
Differences between EcoRI nonspecific and "star" sequence complexes revealed by osmotic stress.
N. Sidorova (2004)
TNF-α-induced increase in intestinal epithelial tight junction permeability requires NF-κB activation
T. Ma (2004)
Detection of sRNA-mRNA interactions by electrophoretic mobility shift assay.
Teppei Morita (2012)
Identification of ligand-receptor binding affinity using AlGaN/GaN high electron mobility transistors and binding-site models
Y. Wang (2013)
Identification of Proteins Interacting with Single Nucleotide Polymorphisms (SNPs) by DNA Pull-Down Assay.
B. Singh (2019)
Transcription factor beacons for the quantitative detection of DNA binding activity.
Alexis Vallée-Bélisle (2011)
Identification of transcription factors controlling the expression of paclitaxel biosynthesis genes in cambial meristematic cells of Taxus cuspidata
Zejun Yan (2013)
Mutational analysis of the transcriptional regulator GcvA: amino acids important for activation, repression, and DNA binding.
A. Jourdan (1998)
Regulators of dormancy/viability of Mycobacterium tuberculosis inside the human macrophages
M. M. Botha (2012)
Mobility Shift DNA‐Binding Assay Using Gel Electrophoresis
S. Buratowski (2001)
Prolactin induction of the alpha 2-Macroglobulin gene in rat ovarian granulosa cells: stat 5 activation and binding to the interleukin-6 response element.
M. Dajee (1996)
Regulation of NF-κB/Rel by IκB is essential for ascidian notochord formation
N. Kawai (2005)
Detection of DNA damage‐recognition proteins using the band‐shift assay and Southwestern hybridization
M. Protic (1993)
Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements
D. Shore (1987)
Discovery of Cellular Regulation by Protein Degradation
A. Varshavsky (2008)
Removing water from an EcoRI-noncognate DNA complex with osmotic stress.
N. Sidorova (1999)
Salt stress alters A/T-rich DNA-binding factor interactions within the phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum
J. Cushman (2004)
Regulation of the expression of proenkephalin mRNA in bovine adrenal chromaffin cells: Role of proto-oncogenes
E. Mar (1992)
In vitro binding of several cell-specific and ubiquitous nuclear proteins to the GT-I motif of the SV40 enhancer.
J. H. Xiao (1987)
An inducible transcription factor activates expression of human immunodeficiency virus in T cells
G. Nabel (1987)
Ethylation interference and X-ray crystallography identify similar interactions between 434 repressor and operator
F. Bushman (1985)
See more
Semantic Scholar Logo Some data provided by SemanticScholar