Melanie Ohi

Structural and Functional Studies of Large Macromolecular Complexes

Proteins carry out most cellular processes as members of dynamic multi-protein assemblies. Although progress has been made cataloging the constituents of specific complexes, we have only limited knowledge of how proteins assemble into macromolecular machines and how these machines perform their cellular functions. We are using yeast genetics, biochemistry and single particle cryo-electron microscopy (EM) to explore the structural and functional organization of complexes involved in pre-mRNA splicing and complexes involved in the protein ubiquitination pathway.

Single Particle Cryo-Electron Microscopy. Single particle cryo-EM is a powerful technique for determining the structures of large, dynamic complexes that are too difficult to crystallize. In this structural approach, purified complexes are applied to grids covered with holey carbon film and quickly frozen by plunging the grids into liquid ethane. The rapid freezing prevents water from forming ice crystals and embeds the molecules in a layer of vitrified (or amorphous) ice preserving the specimen in a near-native environment. Images of the preserved particles are taken using an electron microscope. Digital image processing methods are then used to produce three-dimensional (3D) models from the images of particles trapped in vitrified ice.

The Spliceosome. Although the human genome contains ~25,000 genes, it is estimated that we make over 90,000 proteins. The disparity between our genome and our proteome can be explained by the activity of the spliceosome, a large macromolecular machine composed of RNA and protein components. This complex catalyzes the excision of non-coding introns from a pre-messenger RNA (pre-mRNA) to create a mature message (mRNA). Although the composition of the spliceosome is known, it remains a mystery how this dynamic machine functions. To understand spliceosome function and regulation it will be essential to develop 3D pictures of how the numerous spliceosomal proteins and RNA components organize into one machine. A number of projects in the lab focus on the functional and structural characterization of stable spliceosomal complexes isolated from the fission yeast S. pombe.

The Anaphase Promoting Complex or Cyclosome (APC/C). Ubiquitin (Ub) becomes covalently attached to substrate proteins via an enzyme cascade consisting of activating (E1), conjugating (E2), and ligating (E3) enzymes. E3 ubiquitin ligases vary widely in size, composition, and enzymology reflecting their regulatory role in substrate recognition. The APC/C is an ubiquitin ligase composed of 13 polypeptides that facilitates the transfer of Ub from E2s to specific substrates. The APC/C is regulated at different stages of the cell cycle by both phosphorylation events and by the binding of specific activators or inhibitors to the core complex. Projects in the lab focus on structurally characterizing the APC/C purified from different stages of the cell cycle.

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