Manipulation of chromatin structure is closely linked to the process of gene regulation. The core component of chromatin, the nucleosome, consists of approximately 147 base pairs of DNA wrapped around a protein core consisting of two histone H2A-H2B dimers and a histone H3-H4 tetramer. Chromatin structure can be altered through the action of a diverse assortment of proteins and protein complexes including ATP-dependent remodelling enzymes related to the yeast Snf2 protein. A major factor limiting insight into ATP- dependent chromatin remodelling enzymes is a lack of structural understanding. The Saccharomyces cerevisiae
Snf2 family members Fun30 (Function Unknown Now 30) and Chd1 function as homodimers and monomers respectively and represent tractable systems for structural investigation in comparison to multi-subunit complexes. Here systems are described for expression of Fun30 and Chd1 protein fragments from Saccharomyces cerevisiae
and the thermophile Chaetomium Thermophilium
. Conditions were established to express 6 fragments of these proteins in mg quantities. The ability of these fragments to form stable complexes with nucleosomes was also assessed. The formation of stable complexes between Fun30 and nucleosomes indicated the potential for further structural characterisation especially using cryo electron microscopy.
|Date of Award||2016|
|Supervisor||Tom Owen-Hughes (Supervisor)|