eIF4B and eIF4G jointly stimulate eIF4A ATPase and unwinding activities by modulation of the eIF4A conformational cycle.
Eukaryotic translation initiation factor 4A (eIF4A) is a DEAD-box protein that participates in translation initiation. As an ATP-dependent RNA helicase, it is thought to resolve secondary structure elements from the 5'-untranslated region of mRNAs to enable ribosome scanning. The RNA-stimulated ATPase and ATP-dependent helicase activities of eIF4A are enhanced by auxiliary proteins, but the underlying mechanisms are still largely unknown. Here, we have dissected the effect of eIF4B and eIF4G on eIF4A RNA-dependent ATPase- and RNA helicase activities and on eIF4A conformation. We show for the first time that yeast eIF4B, like its mammalian counterpart, can stimulate RNA unwinding by eIF4A, although it does not affect the eIF4A conformation. The eIF4G middle domain enhances this stimulatory effect and promotes the formation of a closed eIF4A conformation in the presence of ATP and RNA. The closed state of eIF4A has been inferred but has not been observed experimentally before. eIF4B and eIF4G jointly stimulate ATP hydrolysis and RNA unwinding by eIF4A and favor the formation of the closed eIF4A conformer. Our results reveal distinct functions of eIF4B and eIF4G in synergistically stimulating the eIF4A helicase activity in the mRNA scanning process.
University of Muenster, Institute for Physical Chemistry, Corrensstrasse 30, D-48149 Muenster, Germany.
Alexandra Zoi
AZ
Andreou
University of Muenster, Institute for Physical Chemistry, Corrensstrasse 30, D-48149 Muenster, Germany. Electronic address: dagmar.klostermeier@uni-muenster.de.
Dagmar
D
Klostermeier
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D039601:Eukaryotic Initiation Factor-4A / Q000737:chemistry* / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology*
D039603:Eukaryotic Initiation Factor-4G; D039642:Eukaryotic Initiation Factors; C031799:eIF-4B; D039601:Eukaryotic Initiation Factor-4A; D000251:Adenosine Triphosphatases
ATP hydrolysis; DEAD-box helicase; PABP; RNA recognition motif; RRM; conformational change; eIF4A; poly(A)-binding protein; regulation; single-molecule fluorescence resonance energy transfer; single-stranded RNA; smFRET; ssRNA