Date of Award
Master of Science
Biochemistry, Biophysics and Molecular Biology
A common post-translational modification (PTM) of proteins is lysine acetylation. This is an especially ubiquitous PTM in the histones of chromatin, and is important for helping to regulate both structural and mechanistic aspects of chromatin. The fundamental unit of chromatin is called the nucleosome and is made up of DNA that wraps around a histone protein octamer. Protruding from the nucleosome are 10 unstructured “tails” which protrude into the aqueous environment. A number of strategies exist for generating acetylated nucleosomes for the in-vitro study of chromatin including: Purification from eukaryotic organisms, chemical acetylation, amino acid analog incorporation, enzyme mediated acetylation, native chemical ligation of peptides, and enzyme mediated ligation of peptdes. An especially attractive approach is to genetically encode acetyl-lysine residues using nonsense suppression. This strategy has been successfully applied to single sites of histone acetylation. However, because histone acetylation can often occur at multiple sites simultaneously, it is worth while to determine whether this approach could be extended.
The results in this thesis show that recombinantly expressed histone H3 proteins that incorporate up to four sites of lysine acetylation on the histone tail can be produced in good yields. Because the amount of expressed multi-acetylated histone is reduced relative to the wild type, a purification strategy involving affinity purification and ion exchange chromatography was optimized. This expression and purification strategy ultimately generates H3 histone uniformly acetylated at the desired position at levels and purity sufficient to assemble histone octamers. Histone octamers containing four sites of lysine acetylation were assembled into mononucleosomes and enzymatic assays confirmed that this acetylation largely blocks further acetylation by the yeast SAGA acetyltransferase complex.
In the future, multiply acetylated histones may be applied in many ways including two that are of primary interest. Firstly, they can be used to mechanistically probe the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex in yeast which is known to play an active part in the activation of transcription in silent inducible genes. Secondly, they may be used to investigate acetylation’s role in destabilizing nucleosomes.
Young, Isaac, "Introducing multiple sites of acetylation to histone H3 via nonsense suppression" (2016). Graduate Theses and Dissertations. 15850.