Abstract
The eukaryotic translation initiation factor (eIF) 4B promotes the RNA-dependent ATP hydrolysis activity and ATP-dependent RNA helicase activity of eIF4A and eIF4F during translation initiation. eIF4B also helps to organize the assembly of the translational machinery through its interactions with eIF4A, eIF4G, eIF3, the poly(A)-binding protein (PABP), and RNA. Although the function of eIF4B is conserved among plants, animals, and yeast, eIF4B is one of the least conserved of initiation factors at the sequence level. Mammalian eIF4B is a constitutive dimer; however, conflicting reports have suggested that plant eIF4B may exist as a monomer or a dimer. In this study, we show that eIF4B from wheat can form a dimer and we identify the region responsible for its dimerization. Zinc stimulated homodimerization of eIF4B and bound eIF4B with a Kd of 19.7 nM. Zinc increased the activity of the eIF4B C-terminal RNA-binding domain specifically. Zinc promoted the interaction between eIF4B and PABP but not the interaction between eIF4B and eIF4A or eIFiso4G, demonstrating that the effect of zinc was highly specific. The interaction between PABP and eIFiso4G was also stimulated by zinc but required significantly higher levels of zinc. Interestingly zinc abolished the ability of eIFiso4G to compete with eIF4B in binding to their overlapping binding sites in PABP by preferentially promoting the interaction between eIF4B and PABP. Our observations suggest that wheat eIF4B can dimerize but requires zinc. Moreover zinc controls the partner protein selection of PABP such that the interaction with eIF4B is preferred over eIFiso4G.
Highlights
Protein synthesis is an essential step in gene expression, and several global and specific regulatory mechanisms targeting translation have evolved to control the level of proteins
The eIF4 group of initiation factors is important for the recruitment of the ribosome. eIF4F binds to the 5Ј-cap structure of an mRNA and is composed of eIF4E, which is responsible for cap binding, and eIF4G, a modular scaffolding protein that interacts with eIF4E, eIF4A, eIF3, and the poly(A)-binding protein (PABP) (4)
Two eIFiso4G interaction domains are present in PABP: the first maps to a 36-amino acid region encompassing the C-terminal end of RRM1, the linker region, and the N-terminal end of RRM2 to which eIFiso4G binds strongly, whereas the second site maps to RRM3– 4 to which eIFiso4G binds weakly (15)
Summary
Plasmid Construction and Protein Expression—Constructs for the expression of full-length or truncated eIFiso4G, eIF4B, and PABP were described previously (12). Following incubation for 20 min at 4 °C, the poly(A)-Sepharose 4B resin was washed, and the extent of RNA binding was determined by SDS-PAGE. Bound protein was released following the addition of SDS sample buffer to the resin. RNase A was added to the resin containing the bait protein and incubated at 37 °C for 20 min. The prey protein and metal ions were added, and the reaction was incubated at 4 °C for an additional 2 h. Binding experiments were performed using 50 and 150 nM full-length eIF4B protein to which increasing amounts of ZnCl2 and MgCl2 were added individually in separate experiments. His-eIF4B280-527 37 °C for 40 min and stopped by the GST GST-4B 320-360 GST-4B 317-340 GST-4B 320-527 GST-4B 340-527 GST-4B 69-324 GST-4B 69-360
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