Cryo-electron microscopy and functional analysis of the yeast Ccr4-Not deadenylase complex
Final Report Abstract
Ccr4-Not is a 9-subunit protein complex involved in regulating transcription and mRNA stability. It therefore plays a fundamental role in gene expression. The goal of my project was to gain insight into the Ccr4-Not complex through structural and biochemical approaches. I aimed to determine the structure of the intact complex using electron microscopy (EM), define the stoichiometry of the subunits and gain insight into the roles of the exonuclease subunits. From a methods perspective, I hoped to gain hands-on experience with multi-protein complexes and structural techniques. I first established a purification protocol for Ccr4-Not using a novel tag in yeast. This defined some of the protein-protein interactions within the complex. However, due to heterogeneity and low yields, we have been unable to obtain a 3D EM reconstruction and work is ongoing. Biochemical assays revealed insight into the activity of the complex in vitro. I was able to learn cutting-edge techniques in working with multi-protein complexes, and in electron microscopy and single particle reconstruction.
Publications
- Cytoplasmic deadenylation: regulation of mRNA fate. Biochem Soc Trans. 2010 Dec;38(6):1531-6
Wiederhold K, Passmore LA
(See online at https://doi.org/10.1042/BST0381531)
