This proposal aims to determine the role of torsional stress in chromatin structure and transcription. One set of experiments will determine whether or not nucleosomes are displaced during transcription. The next set of experiments will develop synthetic topological domains using a plasmid that will be divided into two domains constrained by lac repressor:operator complexes. The synthetic domains can be formed into a single domain instead of two independent domains by the substitution of a mutant lac repressor which does not tetramerize. Additional experiments will determine whether torsional stress induced in the molecule by transcription is sufficient to reorganize chromatin, or if chromatin reorganization takes place only through direct contact with the polymerase complex. An analysis of nucleosome architecture in transcribed and non-transcribed regions will be carried out, and the rate of transcription will be determined in each case.
