Characterization of the ATF/CREB site and its complex with GCN4

Abstract

We have studied DNA minicircles containing the ATF/CREB binding site for GCN4 by using a combination of cyclization kinetics experiments and Monte Carlo simulations. Cyclization rates were determined with and without GCN4 for DNA constructs containing the ATF/CREB site separated from a phased A-tract multimer bend by a variable length phasing adaptor. The cyclization results show that GCN4 binding does not significantly change the conformation of the ATF/CREB site, which is intrinsically slightly bent toward the major groove. Monte Carlo simulations quantitate the ATF/CREB site structure as an 8° bend toward the major groove in a coordinate frame near the center of the site. The ATF/CREB site is underwound by 53° relative to the related AP-1 site DNA. The effect of GCN4 binding can be modeled either as a decrease in the local flexibility, corresponding to an estimated 60% increase in the persistence length for the 10-bp binding site, or possibly as a small decrease (1°) in intrinsic bend angle. Our results agree with recent electrophoretic and crystallographic studies and demonstrate that cyclization and simulation can characterize subtle changes in DNA structure and flexibility.