DNA trajectory in the Gal repressosome

The Gal repressosome is a higher-order nucleoprotein complex that represses transcription of the gal operon in Escherichia coli. During the repressosome assembly, a DNA loop is formed by the interaction of two GalR dimers, bound to two spatially separated operators, OE and OI, flanking the gal promoters. Structure-based genetic analysis indicated that GalR homodimers interact directly and form a V-shaped stacked tetramer in repressosome, further stabilized by HU binding to an architecturally critical position on the DNA. In this scheme of GalR tetramerization, the alignment of the operators in the DNA loop could be in either parallel (PL) or antiparallel (AL) mode. As each mode can have two alternative geometries differing in the mutual stacking of the OE- and OI-bound GalR dimers, it is possible to have four different DNA trajectories in the repressosome. Feasibilities of these trajectories were tested by in vitro transcription repression assays, first by isolating GalR mutants with altered operator specificity and then by constructing four different potential loops with mutant GalR heterodimers bound to specifically designed hybrid operators in such a way as to give rise to only one of the four putative trajectories. Results show that OE and OI adopt a mutual antiparallel orientation in an under-twisted DNA loop, consistent with the energetically optimal structural model. In this structure the center of the HU-binding site is located at the apex of the DNA loop. The approach reported here can be used to distinguish between otherwise indistinguishable DNA trajectories in complex nucleoprotein machines.