NSF Award
9813427
Jackson, Vaughn<br/><br/>The main objective of this project is to extend our knowledge of the mechanisms that facilitate transcription through nucleosomes. This will be done by testing the hypothesis that positive stress, which is induced during transcription, causes a disruption of histone interactions within the nucleosome. It is specifically proposed that the H2A,H2B is disrupted from H3,H4, such that the majority of H2A,H2B is released. It is proposed that, in contrast, this increase in positive stress increases the affinity of H3,H4 for the DNA, so that H3,H4 transfer during transcription is minimized. The rate of transcription through nucleosomes is proposed to influence both processes, with NAP1 (a histone deposition factor) maximizing the release of H2A,H2B. This model will be tested by utilizing an in vitro transcription system that includes the following components: T7 RNA polymerase, prokaryotic (E. coli) topoisomerase I and eukaryotic (MSB) topoisomerase I, acetylated or nonacetylated histones and additional agents to regulate the level of positive stress, i.e. polyamines and RNase A. The template is a circular, covalently closed DNA with two T7 promoters immediately upstream of 18 repeats of the 207 bp 5S RNA gene of Lytechinus vareigatus, a sequence that positions nucleosomes. The assay for measuring histone mobility includes a competitor DNA in the reaction mix and measures the topological change in the competitor as well as the amount and type of histone bound, by separating components on sucrose gradients and by gel electrophoresis. Formaldehyde crosslinking will also be used to freeze the state of the transcription complexes, which will facilitate the separation and analysis of the nucleosomes.<br/><br/>Histones are highly basic proteins that bind DNA with extremely high binding energies. This binding is required to facilitate the orderly condensation of the large quantity of DNA that is contained in the nucleus of a cell. The compacted structures that are formed are called nucleosomes. These structures, stabilized by the high histone binding energy, repress nuclear processes such as DNA replication, DNA repair, and transcription initiation or elongation. It is therefore important to understand the cellular mechanisms that disrupt these interactions and allow these processes to occur. This project will study one possible mechanism, the induction of positive stress on DNA, which is caused when the DNA is overwound by RNA polymerase, in the course of copying the information into RNA. This study will provide insight into the nuclear dynamics that allow the genetic information in DNA to be duplicated and copied.
