Doctoral Dissertations
Date of Award
12-1982
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Biomedical Sciences
Major Professor
Donald E. Olins
Committee Members
Julian Preston, Kai Lin Lee, Robert Fujumura, Audrey Stevens
Abstract
This dissertation contains results from the study of nucleosome-HMG protein complexes. The complex was studied by thermal denaturation, electrophoresis, circular dichroism, sedimentation and electron microscopy. Initial studies focused on the formation of complexes when HMG 14 or 17 was added to nucleosome core particles. Discrete complexes were observed by electrophoresis on non-denaturing gels. Titration of nucleosome core particles with HMG 14 or 17 caused the cooperative formation of complexes containing 2 HMG proteins per nucleosome. The cooperative binding was dependent on ionic strength. Further studies, employing nuclease digestion and high resolution gel electrophoresis, located the binding sites of HMG proteins along the nucleosomal DMA. These results, coupled with the titration data, resulted in a model for the binding of HMG proteins to the nucleosome.
The preceding work was followed by extensive characterization of the complex. Thermal denaturation and circular dichroism showed HMG 14 and 17 stabilized the nucleosome core particle. These results argued against a role for HMG protein as a structural protein that loosened chromatin structure prior to transcription. Sedimentation in the analytical ultracentrifuge and electron microscopy both showed no significant structural changes in the nucleosome after HMG binding.
Later studies employed urea as an outside perturbant to see if HMG protein could stabilize nucleosomes against unfolding and to see if partially unfolded nucleosomes bound IMG protein. Thermal denaturation, circular dichroism and electrophoresis showed HMG 14 and 17 did stabilize the nucleosome against unfolding by urea. In addition, it was discovered that compact nucleosome structure is required for specific binding of HMG 14 and 17.
Nucleosomes were crosslinked extensively with formaldehyde to test whether the cooperative binding of HMG proteins required a nucleosome conformational change. This experiment also revealed whether the histone basic tails were required for HMG binding. The results showed a significant conformational change was not required for a shift from cooperative to noncooperative HMG binding and that crosslinking the histone tails did not prevent specific binding of HMG 14 or 17. Nucleosomes were trypsinized to remove histone basic tails then complexed with HMG proteins. Electrophoresis showed that removal of histone basic tails destroyed specific binding of HMG proteins.
Recommended Citation
Paton, Arthur Elliot, "Structure of nucleosome-HMG complexes. " PhD diss., University of Tennessee, 1982.
https://trace.tennessee.edu/utk_graddiss/13305