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 that is mediated by a structural BYDV translation element (BTE) located in the 3'-UTR of its mRNA. eIF4F bound the BTE and a translationally 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 to 164% compared with 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 prevented eIF4F binding to mutant BTE but not WT BTE; 3) BTE mutant-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52-90% to ΔG° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding. Further, the eIF4F bound to the 3' BTE with higher affinity than for either m(7)G cap or tobacco etch virus internal ribosome entry site, suggesting that the 3' BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation.
Highlights
Little is known about Barley yellow dwarf virus (BYDV) protein synthesis initiation mechanisms
Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity correlated well with translation efficiency, whereas the “on” rate of binding did not; 2) other unidentified proteins or small molecules in wheat germ extract prevented eIF4F binding to mutant BYDV translation element (BTE) but not WT BTE; 3) BTE mutanteIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52–90% to ⌬G° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding
We show 1) translation efficiencies correlated well with eIF4F binding; 2) as yet unidentified proteins or small molecules in WGE prevented binding of eIF4F to BTEBF mutant but not WT BTE; 3) kinetics experiments showed that the association rate constants were similar for WT and mutants, indicating stability rather than kinetics correlates with translation; and 4) eIF4F interactions with 3Ј BTE and mutants were both enthalpically and entropically favorable
Summary
Little is known about BYDV protein synthesis initiation mechanisms. Results: eIF4F binding correlates with 3Ј BTE translation efficiency and is enthalpically and entropically favorable. Barley yellow dwarf virus (BYDV) employs a cap-independent mechanism of translation initiation that is mediated by a structural BYDV translation element (BTE) located in the 3-UTR of its mRNA. 2 The abbreviations used are: BYDV, barley yellow dwarf virus; BTE, BYDV translation element; BTEBF, mutant of BTE with a CUAG insert at position 4873; WGE, wheat germ extract; d-WGE, depleted WGE; PABP, poly(A)binding protein; eIF, eukaryotic initiation factor; PK1, pseudoknot 1. This quantitative information, together with the stability measurements and kinetics, determined the nature of interactions and yielded information on the role of eIF4F in the sequential assembly of the initiation complex and its ability to facilitate translation initiation
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