Further Biochemical and Kinetic Characterization of Human Eukaryotic Initiation Factor 4H

Abstract

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 ␤-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.The initiation of protein synthesis in eukaryotes is a complex process involving almost a dozen initiation factors that work in combination to bring the mRNA, the initiating methionyltRNA (Met-tRNA i ), 1 and the 40 S ribosomal subunit together into a 48 S translation complex. This 48 S complex can then associate with the 60 S ribosomal subunit to complete the initiation phase of protein synthesis (for recent reviews on translation initiation, see Refs.

Interactions between eIF4AI and its accessory factors eIF4B and eIF4H

“…This suggests that in vivo, 4B and 4H compete with one another for interaction with and regulation of 4AI. Although 4B and 4H appear to be ubiquitously expressed (Nomura et al 1994;Richter et al 1999), tissue-specific variations in their mRNA levels suggest that individual tissues contain unique ratios of the two proteins. For example, 4H transcript levels are twice those of 4B in the brain, whereas 4B transcripts are circa four times those of 4H in skeletal muscle ).…”

Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation

“…(iii) Pateamine may uncouple the coordinated activities between eIF4A f and eIF4B and͞or eIF4H. eIF4B, and eIF4H stimulate the ATPase (3, 5, 6, 34), RNA-binding (5,34,35), and helicase activities (4, 6, 10, 36) of eIF4A f and eIF4A c . This model would suggest that tight regulation of eIF4A f activity is essential for optimal translation initiation on cap-dependent mRNA templates, as well as on eIF4A-dependent IRESes (Figs.…”

The genomic basis of the Williams – Beuren syndrome

“…This gene is ubiquitously expressed and it is suggested that it is involved in translation initiation and in stabilizing attributes during RNA binding, ATP hydrolysis and RNA duplex unwinding [175].…”