A method has been developed for the analysis of transcript elongation by human RNA polymerase II (pol II) through precisely positioned single nucleosomes. Earlier work suggested that nucleosome traversal by pol II is driven by the loss of one of the H2A/H2B dimers. However, results with the new system show that assembly of histones on some DNA sequences (high-barrier sequences) creates a nearly insurmountable barrier to transcript elongation which can be duplicated with the central H3/H4 tetramer alone. Nucleosomes on high-barrier sequences resist traversal even in the presence of transcription factors and histone chaperones that were effective in facilitating traversal in earlier studies. This project will exploit the single-nucleosome system to achieve a greater understanding of the mechanism of nucleosome traversal. Two important questions will be addressed: How do features of histone structure, including the N-terminal tail segments, affect the nucleosomal barrier? Can novel histone chaperones be identified, particularly among those that target the H3/H4 tetramer, which are effective in facilitating nucleosome traversal?
The genetic material, DNA, is packaged by proteins in the nucleus of the cell. The overall goal of this project is to increase our understanding of the mechanisms through which this packaging affects DNA access by the molecular machinery that reads the genetic information. This project is expected to uncover novel factors which regulate the unfolding of the protein packaging of DNA and thereby control the expression of genetic information. The test tube systems developed in the previous funding period should provide an important resource for investigators studying the molecular basis of gene regulation. This project should also have an important educational impact, since novel findings made in the research will be directly communicated to graduate students through the courses which the P.I. instructs.
