Student Theses and Dissertations

Date of Award

2010

Document Type

Thesis

RU Laboratory

Blobel Laboratory

Keywords

nuclear pore complex, RNA transport, Nup214, Ddx19 binding, leukemogenesis, leukemogenesis, mRNA export

Abstract

In order to gain a deeper understanding of the role of nups in leukemogenesis, and to make sense of the architecture and regulation of the mRNA export machinery at the NPC, I set out to biochemically and structurally characterize Nup214. In this thesis, I present the crystal structure of the human Nup214 N-terminal domain at 1.65 Å resolution. The structure reveals a sevenbladed !-propeller fold followed by a 30-residue C-terminal extended peptide segment (CTE). The CTE folds back onto the !-propeller and binds to its bottom face. Conserved surface patches on the Nup214 NTD reveal putative proteininteraction sites, one of which is crucial for the interaction with Ddx19. Using a comprehensive mutational and biochemical analysis, the interaction between the Nup214 NTD and Ddx19 is dissected. The structure of the Nup214 NTD•Ddx19 in its ADP-bound state at 2.5 Å resolution reveals the molecular basis for the interaction between the two proteins. A conserved residue of Ddx19 is shown to be crucial for complex formation in vitro and in vivo. Strikingly, the interaction surfaces exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. Ddx19 is shown to bind RNA only in its ATP-bound state, and the binding of RNA and the Nup214 NTD is mutually exclusive. Finally, I speculate that Nup214 is the ATP-exchange factor for Ddx19, and propose the Ddx19 ATPase cycle as the terminal step in mRNA export.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/351

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