| DNA bending may have important consequences for transcription initiation. When the DNA is bent, it can change protein-DNA and protein-protein contacts. The primary goal of this research was to understand the DNA bending patterns and properties associated with the binding of E. coli RNA polymerase to promoter DNA and subsequent conversion to an elongation complex. Footprinting, microscopy and topology studies from several laboratories suggest that DNA is bent or wrapped around E. coli RNA polymerase during transcription initiation and elongation. However, the direction of bending and even the question of whether the observed bending is due to flexibility or static curvature have not been resolved.; We have addressed this question using DNA fragments designed for cyclization kinetics and minicircle binding experiments. A set of DNA constructs containing sequence directed bends (A-tracts) phased in three different orientations against the lac UV5 promoter have been synthesized. T4 DNA ligase mediated cyclization is used to form minicircles with and without polymerase. DNA molecules whose ends are brought together and properly aligned by bending cyclize more rapidly then unbent DNA. Surprisingly, for RNAP-bound DNA, we find relatively little phase dependency of cyclization, and generally decreased cyclization efficiency. These results suggest that if a conformational change is induced by RNAP it is localized and absorbed by the polymerase, otherwise enhanced cyclization would have been observed for at least one of our phased constructs. This lack of phasing suggests an induced bend of 0°, 180°, or 360°. We report a constant polymerase-induced ΔLk = −1.1 to −1.4 for all of the binary and ternary complexes, which excludes the possibility of a 180° or a 360° bend. Previous topological work in this field yielded similar results. Based on our findings, we propose an overall bend angle of ∼0° (the DNA enters and exits RNAP at the same angle) with the RNAP accommodating all of the topological unwinding with little effect on the geometry of the DNA distal to the protein. |
