Date of Degree

5-2018

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Dixie J. Goss

Committee Members

Frida E. Kleiman

Diana P. Bratu

Ruth E. Stark

Ruben L. Gonzalez

Subject Categories

Biochemistry | Bioinformatics | Biophysics | Molecular Biology | Other Biochemistry, Biophysics, and Structural Biology | Plant Biology | Structural Biology | Virology

Keywords

plant virus, RNA‐protein interaction, RNA structure, eukaryotic translation initiation, translation initiation factor, RNA untranslated region

Abstract

5' m7GpppN cap and the 3' poly adenosine (A) tail of eukaryotic mRNAs are key elements for recruiting translation initiation machinery in canonical translation initiation. Unlike host mRNAs, many viruses lack these elements and yet they are translated efficiently. Plant viruses, in particular, have complex structures within their untranslated regions (UTR) that allow them to bypass some cellular translation control steps. In Maize necrotic streak virus (MNeSV) 3' UTR, an I-Shaped RNA Structure (ISS) has been reported to mediate the virus translation initiation progress. 3’ ISS binding with eIF4F has been shown to facilitate translation. 5’ -3’ kissing loop interaction was required for optimal translation. However, details of ISS mediation on translation initiation are still not well understood. In our study, fluorescence anisotropy techniques were applied to study the binding of 3' ISS with eIFs. eIF4A-eIF4B complex was found to increase the binding affinity of eIF4F with 3'ISS by four-fold (from KD=~173±34 nM to KD=~48±11 nM). Pre-steady state analysis demonstrated that eIF4A-eIF4B complex increased association rate and decreased dissociation rate. The enhanced binding affinity was not caused by helicase activity of the eIF4A-eIF4B complex. Besides, our study also suggested that eIF4F could promote binding of 3’ ISS with 5’ UTR, which resembles the long distance kissing loop interaction. Presence of 5’ UTR would not affect 3’ ISS-eIF4F complex’s moderate binding with the 40S ribosomal subunit.

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