Further biochemical and kinetic characterization of human eukaryotic initiation factor 4H

A cDNA encoding human eukaryotic initiation factor (eIF) 4H was subcloned i nto a bacterial expression plasmid for purification of recombinant protein. Recombinant human eIF4H (heIF4H) was purified to greater than 95% homogene ity and shown to have similar physical characteristics to eIF4H purified fr om rabbit reticulocyte lysate as described previously. Functional studies h ave revealed that recombinant heIF4H functions identically to rabbit eIF4H in stimulating protein synthesis, and the ATP hydrolysis and helicase activ ities of eIF4A, More detailed enzymatic studies revealed that eIF4H increas es the affinity of eLF4A for RNA by a-fold, but has no effect on the bindin g of ATP by eIF4A, eIF4H stimulates the helicase activity of eIF4A at least 4-fold, and it is postulated that this stimulation occurs through increasi ng the processivity of eIF4A Northern blot analysis shows that eIF4H is exp ressed ubiquitously in human tissues, and displays different levels of expr ession in given tissues relative to eIF4B. Secondary structure analysis of heIF4H by circular dichroism suggest that eIF4H has a mostly beta-sheet str ucture, which appears similar to other RNA recognition motif-containing pro teins. Finally, it is suggested that eIF4H functions in translation initiat ion through protein-protein interactions that possibly stabilize conformati onal changes that occur in eIF4A during RNA binding, ATP hydrolysis, and RN A duplex unwinding.