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Comparative gel electrophoresis measurement of the DNA bend angle induced by the catabolite activator protein
Author(s) -
Zinkel Sandra S.,
Crothers Donald M.
Publication year - 1990
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.360290106
Subject(s) - chemistry , electrophoresis , dna , gel electrophoresis , electrophoretic mobility shift assay , activator (genetics) , molecule , catabolite repression , biophysics , crystallography , microbiology and biotechnology , biochemistry , gene , biology , gene expression , organic chemistry , mutant
We describe a method to determine the magnitude of protein‐induced DNA bends relative to a set of standard A tract bends using comparative gel electrophoresis. The DNA bend of interest was that induced by the catabolite activator protein (CAP), the transcriptional activator protein of the lac operon. The set of comparison molecules contained both bends of known magnitude and a bound CAP. The electrophoretic influence of the bound protein was accounted for by placing its binding site at the end of the molecule where its induced bend has little influence. Standard bends at the DNA center were introduced by incorporating 3–9 A 6 tracts at ∼ 10.5 base‐pair phasing. The mobility of these control molecules was compared to the mobility of a test molecule of comparable length containing a central CAP‐induced DNA bend. The CAP bend angle was found to be 5.6 ± 0.3 A tract equivalents, or ∼ 100°, independent of the concentration of the gel used within the range tested. The dependence of gel retardation on DNA end‐to‐end distance was found to break down for A tract bend angles above 120°, corresponding roughly to the angle beyond which the long axis of the molecule is no longer parallel to the end‐to‐end vector. We speculate that this may reflect a switch in the mode of migration of molecules through the gel.