Date of Degree

2-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor(s)

Dixie J. Goss

Committee Members

Lesley Davenport

Frida E. Kleiman

Richard Magliozzo

Mandë Holford

Subject Categories

Biochemistry | Biophysics | Molecular Biology

Keywords

Protein RNA Interaction, Virus RNA Translation

Abstract

Barley Yellow Dwarf Virus (BYDV) lacks a 5’ (7-methyl guanosine) cap as well as a 3’poly A tail. Like many plant viruses, BYDV contains a cap independent translation element (CITE) in the 3’ untranslated region of the viral mRNA. BTE (Barley Yellow Dwarf Virus like cap-independent translation element) is one of the well characterized CITEs. BTE mediated translation primarily depends on eukaryotic initiation factor eIF4G. BTE binds to eIF4G; however, the details of BTE initiated translation are still unclear. Three eIF4G deletion mutants with different domain organization were used to investigate BTE interaction with eIF4G: eIF4G601-1196 is the eIF4G fragment containing amino acid residues from 601 to 1196, including binding domains for eIF4E, central eIF4A, eIF4B and the possible BTE binding region; eIF4G601-1488 is a longer fragment with one additional C-terminal eIF4A binding domain; eIF4G742-1196 is a shorter deletion mutant lacking the eIF4E binding sequence. eIF4G601-1196 binds BTE as efficiently as wild type eIF4G and supports translation. Translation initiation factor eIF4A and eIF4B with ATP (helicase complex) stimulate eIF4G601-1196 binding with BTE but not eIF4G601-1488, suggesting that the helicase complex function relies on the eIF4G central eIF4A binding domain, not the C-terminal eIF4A binding domain. This suggests that, similar to human eIF4G, the wheat eIF4A binding site may serve a regulatory role. eIF4E, upon binding with eIF4G mutants which have the eIF4E binding region, significantly increases the binding to BTE. This indicates that the smaller eIF4G mutant has a more flexible structure that can be positively influenced by eIF4E.

 
 

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