Degree Type

Dissertation

Date of Award

2001

Degree Name

Doctor of Philosophy

Department

Plant Pathology and Microbiology

Major

Genetics

First Advisor

W. Allen Miller

Abstract

Barley yellow dwarf virus RNA contains a translation element (3 'TE) that confers efficient cap-independent initiation at the 5 proximal AUG. Direct end-labeling of RNAs verified the absence of a 5 ' modification on virion RNA. Thus BYDV differs from related viruses by having neither a genome-linked protein nor a 5 ' cap. To function in the 3' UTR, the 3 'TE must recruit ribosomes and associated translation factors, and communicate with the 5' end of the mRNA where translation initiates. The communication function is mediated by direct base pairing between the 3'TE and the 5' UTR. We propose that protein factors interact with the 3'TE to facilitate initiation and ribosome recruitment. The 3'TE binds to initiation factor eIF4F, demonstrated by using the 3'TE to purify this complex from wheat germ extract. eIF4F is a complex of two factors, cap binding protein eIF4E and adaptor protein eIF4G. Using purified initiation factors, both eIF4E and eIFiso4E bound directly and specifically to the TE, with a Kd≈2*10-7M. Non-functional TE mutants did not interact with eIF4E/iso4E. The binding of initiation factor 4E follows the genetic evidence that the 3' TE can functionally substitute for a cap both in vitro and in vivo. Because the TE is located in the 3' UTR of BYDV, we speculate that formation of the initiation complex may not be required at the 5 ' end, but may be delivered there by additional mechanisms. In plant cells dependent on both a cap and poly(A) tail, additional viral sequence (at most 869 nt) beginning five bases upstream of the 3'TE is required for translation. Previously, we found that the function of this additional sequence can be at least partially replaced by a poly(A) tail, suggesting a similar role. This downstream element, bases 5010 to 5677, is dubbed the poly(A) mimic (PAM). The PAM can also function in conjunction with a 5 ' cap. Analysis of PAM mutants defines three interacting sequences with differing roles in translation. These data show that independent elements in BYDV have evolved to functionally replace the roles of a cap and poly(A) tail in translation of the viral mRNA.

DOI

https://doi.org/10.31274/rtd-180813-1834

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu

Copyright Owner

Edwards M. Allen

Language

en

Proquest ID

AAI3034167

File Format

application/pdf

File Size

191 pages

Share

COinS