Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector

Jin Kim, Christian Zwieb, Carl Wu, Sankar Adhya

Research output: Contribution to journalArticlepeer-review

315 Scopus citations

Abstract

The binding of a protein to its specific sequence, borne on a DNA fragment, retards the mobility of the fragment in a characteristic way during gel electrophoresis. If the protein induces bending in the DNA, the contortion can also be monitored by gel electrophoresis, because the amount of retardation of the mobility of the DNA-protein complex is dependent upon the position and the degree of the bend induced in the DNA fragment [Wu and Crothers, Nature 308 (1984) 509-513]. We have constructed a plasmid, pBend2, which can generate a large number of DNA fragments of identical length in which the protein-binding nucleotide sequence is located in circular permutations. The vector contains two identical DNA segments containing 17 restriction sites in a direct repeat spanning a central region containing cloning sites. The protein-binding sequence is inserted at one of these cloning sites. To investigate the functional significance of bending, we have compared, using pBend2, the cAMP · cAMP-receptor protein (CRP)-induced bending of CRP-binding sites found in five different genes of Escherichia coli. We have also shown that the bacteriophage λ OR1 operator DNA is bent when complexed with the CI or Cro repressor of the phage.

Original languageEnglish (US)
Pages (from-to)15-23
Number of pages9
JournalGene
Volume85
Issue number1
DOIs
StatePublished - Dec 21 1989
Externally publishedYes

Keywords

  • Recombinant DNA
  • bacteriophage λ operator
  • gel retardation

ASJC Scopus subject areas

  • Genetics

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