Comparative gel electrophoresis measurement of the DNA bend angle induced by the catabolite activator protein.
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 A6 tracts at approximately 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 approximately 100 degrees, 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 degrees, 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.
Zinkel
S S
SS
Department of Molecular Biophysics, Yale University, New Haven, Connecticut 06512.
Crothers
D M
DM
D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002373:Cyclic AMP Receptor Protein; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation
D002373:Cyclic AMP Receptor Protein; D004247:DNA