Supercoiling facilitates lac operator-repressor-pseudooperator interactions

The binding affinity of the Escherichia coli lactose repressor to operator-containing plasmids was increased by negative supercoiling of the DNA. The increased affinities observed were dependent on the sequence context of the DNA as well as the degree of supercoiling. Dissociation rate constants for...

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Bibliographic Details
Published in:The Journal of biological chemistry 1987-04, Vol.262 (11), p.4943-4946
Main Authors: Whitson, P.A., Hsieh, W.T., Wells, R.D., Matthews, K.S.
Format: Article
Language:English
Subjects:
Biological and medical sciences
DNA, Bacterial - analysis
DNA, Superhelical - analysis
Escherichia coli
Fundamental and applied biological sciences. Psychology
Kinetics
Lac Operon
Molecular and cellular biology
Molecular genetics
Nucleic Acid Conformation
Potassium Chloride - pharmacology
Repressor Proteins - metabolism
Transcription Factors - metabolism
Transcription. Transcription factor. Splicing. Rna processing
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Summary:The binding affinity of the Escherichia coli lactose repressor to operator-containing plasmids was increased by negative supercoiling of the DNA. The increased affinities observed were dependent on the sequence context of the DNA as well as the degree of supercoiling. Dissociation rate constants for plasmids containing a single operator site decreased as a function of the negative supercoil density. However, the presence of pseudooperators in the plasmid DNA in addition to the primary operator sequence resulted in a significant decrease in the operator-plasmid dissociation rate at higher negative supercoil densities. Approximately eight ionic interactions were determined for both the supercoiled plasmids and the linear DNAs examined. These results suggest that the stabilization provided by the topology of supercoiled DNA affects the nonionic component of the protein-DNA interaction. The ability to form a ternary complex of protein with two DNA segments is increased by the presence of multiple operator-like sites on the DNA. Furthermore, supercoiling DNA with multiple operator-like sequences profoundly diminishes the dissociation rate and results in a remarkably stable ternary, presumably looped complex (t1/2 approximately 28 h). These data suggest a critical role in vivo for DNA topology and pseudooperator(s) in transcriptional regulation of the lac operon.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)61135-4