TY - JOUR
T1 - Bending of DNA by gene-regulatory proteins
T2 - construction and use of a DNA bending vector
AU - Kim, Jin
AU - Zwieb, Christian
AU - Wu, Carl
AU - Adhya, Sankar
PY - 1989/12/21
Y1 - 1989/12/21
N2 - 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.
AB - 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.
KW - Recombinant DNA
KW - bacteriophage λ operator
KW - gel retardation
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U2 - 10.1016/0378-1119(89)90459-9
DO - 10.1016/0378-1119(89)90459-9
M3 - Article
C2 - 2533576
AN - SCOPUS:0024814234
VL - 85
SP - 15
EP - 23
JO - Gene
JF - Gene
SN - 0378-1119
IS - 1
ER -