Further biochemical and kinetic characterization of human eukaryotic initiation factor 4H.
A cDNA encoding human eukaryotic initiation factor (eIF) 4H was subcloned into a bacterial expression plasmid for purification of recombinant protein. Recombinant human eIF4H (heIF4H) was purified to greater than 95% homogeneity and shown to have similar physical characteristics to eIF4H purified from rabbit reticulocyte lysate as described previously. Functional studies have revealed that recombinant heIF4H functions identically to rabbit eIF4H in stimulating protein synthesis, and the ATP hydrolysis and helicase activities of eIF4A. More detailed enzymatic studies revealed that eIF4H increases the affinity of eIF4A for RNA by 2-fold, but has no effect on the binding of ATP by eIF4A. eIF4H stimulates the helicase activity of eIF4A at least 4-fold, and it is postulated that this stimulation occurs through increasing the processivity of eIF4A. Northern blot analysis shows that eIF4H is expressed ubiquitously in human tissues, and displays different levels of expression in given tissues relative to eIF4B. Secondary structure analysis of heIF4H by circular dichroism suggest that eIF4H has a mostly beta-sheet structure, which appears similar to other RNA recognition motif-containing proteins. Finally, it is suggested that eIF4H functions in translation initiation through protein-protein interactions that possibly stabilize conformational changes that occur in eIF4A during RNA binding, ATP hydrolysis, and RNA duplex unwinding.
Richter
N J
NJ
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.
Rogers
G W
GW
Hensold
J O
JO
Merrick
W C
WC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D039642:Eukaryotic Initiation Factors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism
C111495:EIF4H protein, human; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins