Single-molecule studies of the eukaryotic translation initiation factor 4A: helicase activity, conformational dynamics and function regulation
The PI3K/Akt/mTOR pathway regulates several cellular functions, including cellular proliferation, growth, and survival. The PI3K/Akt/mTOR pathway converges on the eukaryotic translation initiation factor 4F (eIF4F), making it an attractive molecular target for anti-cancer therapies. As a subunit of eIF4F, eIF4A is known to facilitate binding and scanning of the ribosome by unwinding secondary structures in the 5' untranslated region (UTR) of mRNAs. However, the molecular mechanisms of eIF4A activity have remained elusive. Single-molecule Fluorescence Resonance Energy Transfer (sm-FRET) can probe structural changes and interactions of biological systems in real time, which cannot be observed using bulk techniques. First we directly observe and quantify the helicase activity of eIF4A in the presence of the ancillary RNA-binding factor eIF4H using sm-FRET. We show that eIF4H can significantly enhance the helicase activity of eIF4A by strongly binding both to loop structures within the RNA substrate as well as to eIF4A. Electrophoretic mobility shift assay (EMSA) shows that eIF4H binds to the amino-terminal domain (NTD) but not to the carboxyterminal domain (CTD) of eIF4A. In the presence of ATP, the eIF4A/eIF4H complex exhibits rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. Second, we directly probe the conformational dynamics of eIF4A, in real time, using smFRET. We demonstrate that the eIF4A in the presence of eIF4H can repetitively unwind the RNA hairpin substrate by transitioning between an "open" and a "closed" conformation using the energy from ATP hydrolysis. Upon binding of an RNA hairpin and ATP, which is mediated by eIF4H, eIF4A adopts a closed conformation; after ATP hydrolysis, eIF4A returns to the open conformation and the RNA duplex is completely unwound. Then the eIF4A releases the RNA and the hairpin is quickly reformed. Third, we find that RNA aptamer and the small molecule hippuristanol can inhibit the binding to the RNA substrate or the helicase activity of the eIF4A/eIF4H complex respectively. The RNA aptamer can directly compete with an RNA hairpin for binding to both eIF4A and eIF4H. Hippuristanol inhibits helicase activity by blocking the conformational change of eIF4A.
Thesis (Ph.D.)--Boston University