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Detection of conformational and net charge differences in DNA‐protein complexes by quantitative electrophoresis on polyacrylamitie‐agarose copolymer gels
Author(s) -
Orbán Lászó,
Chrambach Andreas,
Zwieb Christian,
Adhya Sankar L.
Publication year - 1991
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150120602
Subject(s) - lac repressor , dna , repressor , base pair , chemistry , agarose gel electrophoresis , operon , crystallography , microbiology and biotechnology , stereochemistry , lac operon , escherichia coli , biology , plasmid , biochemistry , gene , gene expression
The Galactosidase repressor (GalR) of Escherichia coli modulates the expression of the gal operon by binding to two DNA operators, O E and O 1 . The O E and O 1 elements are 16 by pallindromic DNA sequences, differing in four of the base pairs (Fig. 1). O E and O 1 DNA fragments, both free and complexed with repressor, were analyzed by “quantitative gel electrophoresis”. By the criteria of that method, applied to the linear Ferguson plots of both DNA fragments and the linear ranges of those of the DNA‐GalR complexes, it was shown that the apparent size of DNA increases upon repressor binding. Moreover, this size increase is greater for the complex with the O 1 operator than for the complex with the O E operator in the case that GalR is located in the center of a 155 bp DNA fragment. This is not the case when GalR is located in a peripheral position. By contrast with their size differences, the centrally located GalR‐ O 1 and GalR‐ O E complexes appear to possess indistinguishable net surface charge densities as judged from the intercepts with the mobility axis. The larger size of the complex with centrally located O 1 fragment, as compared with that bearing the O E fragment, is interpreted as being due to bending of the DNA‐protein complex, since an authentically bent fragment of a plasmid with bent upstream activator sequence also exhibits a larger slope of the Ferguson plot, and thus the larger size, than predicted on the basis of its DNA chain length (bp). Thus, the apparently larger size of the GalR‐ O 1 complex at the center of DNA, compared to that of the GalR‐ O E complex, supports the previous conclusion that the Gal repressor induces a larger degree of bending in O 1 DNA than in O E DNA. DNA fragments bearing O E and O 1 when complexed with the Gal repressor show convex Ferguson plots at polyacrylamide concentrations below that of gelation and in the presence of 0.5 % agarose, while the plots of the corresponding free 155 bp DNA fragments are linear in that range. The curvature presumably reflects the size increase concomitant with complex formation, since DNA larger than 155 bp also exhibits that curvature on polyacrylamide stabilized by nonrestrictive agarose gels.