Date of Degree

6-2014

Document Type

Dissertation

Degree Name

Ph.D.

Program

Chemistry

Advisor(s)

Dixie J. Goss

Subject Categories

Biophysics | Chemistry | Molecular Biology

Keywords

Barley Yellow Dwarf Virus, Fluorescence, Protein synthesis, Translation initiation

Abstract

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley Yellow Dwarf Virus (BYDV) employs a cap-independent mechanism of translation initiation which is mediated by a structural element BTE (BYDV translation element) located in the 3’ UTR of its mRNA. eIF4F bound the BTE and a translational inactive mutant with high affinity; thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5-164% compared to WT were studied. Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity (complex stability) correlated well with translation efficiency, whereas the “on” rate of binding did not. 2) other unidentified proteins or small molecules in wheat germ extract (WGE) prevented eIF4F binding to mutant BTE but not WT BTE. 3) BTE mutants-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52-90% to ΔG° at 25°C suggesting hydrogen bonding contributes to stability and 4) in contrast to cap-dependent and tobacco etch virus (TEV) Internal Ribosome Entry Site (IRES) interaction with eIF4F, PABP did not increase eIF4F binding. Further, the eIF4F bound to the 3’ BTE with higher affinity than for either m7G cap or TEV IRES, suggesting that the 3’ v BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation.

In another project, we studied the interaction of a deletion mutant of wheat eukaryotic initiation factor 4B (eIF4B320-527) with zinc using the biophysical technique of circular dichroism. eIF4B is suspected to be a metalloprotein and it is known that zinc stimulates eIF4B self-association at physiological concentrations . It was found that in the presence of zinc there is significant change in the secondary structure of eIF4B320-527. There was approximately a 70% change in the presence of 500 μM zinc and around 38% change in the presence of 500 μM magnesium in alpha content as compared to native protein. There was a change observed in beta sheet content. The changes in secondary structure caused by zinc may be the one of the causes for the eIF4B self-association or enhanced eIF4B-PABP interaction. These results enhance our understanding of the molecular mechanisms by which cell controls translation initiation which is the rate limiting step of cellular protein synthesis.

 
 

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