DNA sequences containing short adenine tracts are intrinsically curved and play a role in transcriptional regulation. Despite many high-resolution NMR and x-ray studies, the origins of curvature remain disputed. Long-range restraints provided by 85 residual dipolar couplings were measured for a DNA decamer containing an adenine (A) 4-tract and used to refine the structure. The overall bend in the molecule is a result of in-phase negative roll in the A-tract and positive roll at its 5 junction, as well as positive and negative tilt inside the A-tract and near its junctions. The bend magnitude and direction obtained from NMR structures is 9.0°into the minor groove in a coordinate frame located at the third AT base pair. We evaluated long-range and wedge models for DNA curvature and concluded that our data for A-tract curvature are best explained by a ''delocalized bend'' model. The global bend magnitude and direction of the NMR structure are in excellent agreement with the junction model parameters used to rationalize gel electrophoretic data and with preliminary results of a cyclization kinetics assay from our laboratory. I t has been known for Ϸ20 years that DNA molecules containing four to six consecutive adenine-thymine base pairs exhibit intrinsic curvature (1). This curvature can play a significant role in transcriptional activation by affecting promoter geometry. Many transcriptional activators are DNA-bending proteins that can either recognize DNA bases (direct recognition) or specific DNA properties such as flexibility (indirect recognition) (2). Escherichia coli promoters frequently contain an adenine (A)-tract region, mostly centered around the Ϫ44 region, which when mutated has been shown to reduce transcription (3). In some cases, substitution of an entire promoter region by properly curved DNA can activate in vitro transcription (4, 5). More recent work indicates that these sequences function as upstream recognition elements (UP elements), the curvatures of which play an unknown role (6). In addition, HIV-1 reverse transcriptase termination of the (ϩ) strand DNA synthesis is thought to occur because of minor groove compression of duplex DNA caused by the A-tracts (7,8). Understanding A-tract geometry can therefore play an important role in our understanding of gene expression. Despite many structural efforts, no consensus about the stereochemical origins of the bend has yet emerged. A-tract molecules studied by x-ray crystallography are all bent in directions orthogonal to that established by gel and solution studies (1). Solution NMR, which is not prone to the artifacts of the crystal environment, has only recently been able to provide the long-range restraints necessary to determine global DNA properties (9). We present application of dipolar couplings to determination of DNA bending of an A 4 -tract and discuss implications for DNA-bending models and stereochemical origins of curvature. Because reliable values for the magnitude and direction of DNA bends can be obtained by cyclization kinetics (10...

Popular programs for characterizing DNA structure include Curves 5.1 (Lavery, R. and Sklenar, H., J. Biomol. Struct. Dyn. 6, 63-91, 1988; Lavery, R. and Sklenar, H., J. Biomol. Struct. Dyn. 6, 655-67, 1989) and Freehelix98 (Dickerson, R. E., Nucleic Acids Res. 26, 1906-1926, 1998), along with the more recent 3DNA (X. J. Lu, Z. Shakked and W. K. Olson., J. Mol. Biol. 300, 819-840 (2000). Given input of structural coordinates, all of these programs return values of the local helical parameters, such as roll, tilt, twist, etc. The first two programs also provide characterization of global curvature. Madbend (Strahs, D. and Schlick, T., J. Mol. Biol. 301, 643-663, 2000), a program that computes global curvature from local roll, tilt, and twist parameters, can be applied to the output of all three structural programs. We have compared the curvature predicted by the three programs with and without the use of Madbend. Global bend magnitudes and directions as well as values of helical kinks were calculated for four high-resolution DNA structures and four model DNA helices. Global curvature determined by Curves 5.1 without Madbend was found to differ from values obtained using Freehelix98 with or without Madbend or 3DNA and Curves 5.1 with Madbend. Using model helices, this difference was attributed the fact that Curves 5.1 is the only program sensitive to changes in axial displacement, such as shift and slide. Madbend produced robust values of bend magnitude and direction, and displayed little sensitivity to axis displacement or the source of local helical parameters. Madbend also appears to be the method of choice for bending comparisons of high-resolution structures with results from cyclization kinetics, a method that measures DNA curvature as a vectorial sum of local roll and tilt angles.