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

doi:10.1016/0378-1119(89)90459-9

Gene

Volume 85, Issue 1, 21 December 1989, Pages 15-23

https://doi.org/10.1016/0378-1119(89)90459-9 Get rights and content

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 O_R1 operator DNA is bent when complexed with the CI or Cro repressor of the phage.

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Citation Excerpt :
A frequent feature of minor groove binding is the induction of sharp DNA bends. Bend angles can be determined using the circular permutation assay (Kim et al., 1989) and also by crystallography, although the latter can be subject to uncertainty as a result of crystal packing forces enhancing or reducing the DNA bend (Masse et al., 2002; Murphy et al., 1999). Solution methods of measuring bend angles have obvious advantages, in particular NMR studies of protein-DNA complexes (Love et al., 1995; Dow et al., 2000; Masse et al., 2002) and in this context fluorescence resonance energy transfer (FRET) is a valuable tool.
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Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector

Abstract

J. Bol. Biol.

J. Mol. Biol.

Cell

Cell

J. Mol. Biol.

J. Biol. Chem.

Cell

Cell

J. Mol. Biol.

Multipartite genetic control elements: communication by DNA loop

Annu. Rev. Genet.

DNA bending induced by the catabolite activator protein allows ring formation of a 144-bp DNA

J. Biomol. Struct. Dyn.

Mutations that alter the DNA-sequence specificity of the catabolite gene activator protein of E. coli

Nature

How the cyclic AMP receptor protein of Escherichia coli works

Mutant of λ phage repressor with a specific defect in its positive control function

Interaction of spatially separated protein-DNA complexes for control of gene expression: operator conversions

Mutants of the catabolite activator protein of Escherichia coli that are specifically deficient in the gene-activation function

Static and initiator proteinenhanced bending of DNA at a replication origin

Science

Variations of intramolecular ligation rates allow the detection of protein-induced bends in DNA

EMBO J.