Duplex unwinding and ATPase activities of the DEAD-box helicase eIF4A are coupled by eIF4G and eIF4B

By Ali R. Özeş, K. Feoktistova, Brian C. Avanzino, C. Fraser. 2011

123 citations recorded by the source corpus.

Eukaryotic initiation factor 4A (eIF4A) is a DEAD-box helicase that stimulates translation initiation by unwinding mRNA secondary structure. The accessory proteins, eIF4G, eIF4B, and eIF4H enhance the duplex unwinding activity of eIF4A, but the extent to which they modulate eIF4A activity is poorly understood. Here, we use real time fluorescence assays to determine the kinetic parameters of duplex unwinding and ATP hydrolysis by these initiation factors. To ensure efficient duplex unwinding, eIF4B and eIF4G cooperatively activate the duplex unwinding activity of eIF4A. Our data reveal that eIF4H is much less efficient at stimulating eIF4A unwinding activity than eIF4B, implying that eIF4H is not able to completely substitute for eIF4B in duplex unwinding. By monitoring unwinding and ATPase assays using identical conditions, we demonstrate that eIF4B couples the ATP hydrolysis cycle of eIF4A with strand separation, thereby minimizing non-productive unwinding events. Using duplex substrates with altered GC contents, but with similar predicted thermal stabilities, we further show that the rate of formation of productive unwinding complexes is strongly influenced by the local stability per base pair in addition to the stability of the entire duplex. This finding explains how a change in the GC content of a hairpin while maintaining overall predicted thermal stability is able to influence translation initiation.

Subfields: Molecular Biology

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In Molecular Biology

1. M. Sokabe
2. C. Fraser
3. A. Hinnebusch
4. K. Feoktistova
5. Enkhee Tuvshintogs
6. A. Do
7. I. S. Fernández
8. D. Ron

Citations in the corpus

• The scanning mechanism of eukaryotic translation initiation. (2014, background): “For the latter activity, eIF4H is less effective than meIF4B (156), consistent with its inability to replace meIF4B in promoting 48S PIC assembly on an mRNA with structured 5′UTR.”
• Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity (2013, methodology): “Unwinding reactions are performed in a 50-μL cuvette (Starna) by using a Fluorolog-3 spectrofluorometer (Horiba) as previously described (21).”
• Toward a Kinetic Understanding of Eukaryotic Translation. (2018, methodology): “To enable 43S PIC binding, a secondary structure near the cap-bound eIF4F complex is unwound by the helicase activity of its eIF4A subunit, together with eIF4B (or eIF4H) (Rozen et al. 1990; Rogers et al. 2001; Nielsen et al. 2011; Özeş et al. 2011; Harms et al. 2014; see Merrick and Pavitt 2018).”