Eukaryotic initiation factor (eIF) 4A is the prototypic member of the DEAD box family of proteins and has been proposed to act as an RNA helicase to unwind secondary structure in the 5-untranslated region of eukaryotic mRNAs. Previous studies have shown that the RNA helicase activity of eIF4A is dependent on the presence of a second initiation factor, eIF4B. In this report, eIF4A has been demonstrated to function independently of eIF4B as an ATP-dependent RNA helicase. The biochemical and kinetic properties of this activity were examined. By using a family of RNA duplexes with an unstructured single-stranded region followed by a duplex region of increasing length and stability, it was observed that the initial rate of duplex unwinding decreased with increasing stability of the duplex. Furthermore, the maximum amount of duplex unwound also decreased with increasing stability. Results suggest that eIF4A acts in a non-processive manner. eIF4B and eIF4H were shown to stimulate the helicase activity of eIF4A, allowing eIF4A to unwind longer, more stable duplexes with both an increase in initial rate and maximum amount of duplex unwound. A simple kinetic model is proposed to explain the mechanism by which eIF4A unwinds RNA duplex structures in an ATP-dependent manner.Initiation of protein synthesis in mammalian systems is a complex process in which the 40 S and 60 S ribosomal subunits are joined with mRNA and initiator methionyl-tRNA (MettRNA i ) 1 to form a translationally competent 80 S initiation complex (for reviews on translation, see Refs. 1-3). Prior to the formation of an 80 S complex, a 48 S pre-initiation complex composed of the mRNA, 40 S ribosomal subunit, initiator methionyl-tRNA, and several initiation factors (eIFs) must be formed. The first major event in the formation of this 48 S complex is the binding of eIF2, GTP, and Met-tRNA i to the 40 S ribosomal subunit, which is facilitated by eIF1A (1). The second major event is the binding of mRNA to this 40 S ribosomal subunit. This is a key regulatory step in the initiation of protein synthesis and involves eIF3, eIF4A, eIF4B, eIF4F, and possibly eIF4H, a novel initiation factor demonstrated to interact with RNA (1, 3, 4).Eukaryotic mRNAs are typically recognized via their 7-methylguanosine (m 7 G) cap structure by eIF4E, the small subunit of eIF4F (5). eIF4F also contains a large subunit (170 kDa), eIF4G, and a subunit that is eIF4A (thus eIF4A may exist as part of eIF4F or in free form) (6). Following binding of the eIF4F to the 5Ј end of the mRNA, initiation factors eIF4A and eIF4B interact with the mRNA and disrupt secondary/ tertiary structure existing in the 5Ј-untranslated region (UTR) through the helicase activity of eIF4A (7-9). This facilitates the binding of the 40 S ribosomal subunit to the 5Ј end of the mRNA and allows for subsequent scanning by the 40 S subunit to the initiating AUG codon and correct placement of the MettRNA i . Although the general sequence of events is known, the molecular mechanism by which these steps take place is still...

Eukaryotic initiation factor (eIF) 4A is a DEAD box RNA helicase that works in conjunction with eIF4B, eIF4H, or as a subunit of eIF4F to unwind secondary structure in the 5-untranslated region of mRNA, which facilitates binding of the mRNA to the 40 S ribosomal subunit. This study demonstrates how the helicase activity of eIF4A is modulated by eIF4B, eIF4H, or as a subunit of eIF4F. Results indicate that a linear relationship exists between the initial rate or amplitude of unwinding and duplex stability for all factor combinations tested. eIF4F, like eIF4A, behaves as a non-processive helicase. Either eIF4B or eIF4H stimulated the initial rate and amplitude of eIF4A-dependent duplex unwinding, and the magnitude of stimulation is dependent on duplex stability. Furthermore, eIF4A (or eIF4F) becomes a slightly processive helicase in the presence of eIF4B or eIF4H. All combinations of factors tested indicate that the rate of duplex unwinding is equivalent in the 5 3 3 and 3 3 5 directions. However, the optimal rate of unwinding was dependent on the length of the single-stranded region of the substrate when different combinations of factors were used. The combinations of eIF4A, eIF4A ؉ eIF4B, eIF4A ؉ eIF4H, and eIF4F showed differences in their ability to unwind chemically modified duplexes. A simple model of how eIF4B or eIF4H affects the duplex unwinding mechanism of eIF4A is proposed. Eukaryotic initiation factor (eIF)1 4A is the prototypic member of the DEAD box family of ATP-dependent RNA helicases (1). DEAD box (and related DEXH box) proteins share eight highly conserved amino acid sequence motifs and are involved in almost all aspects of RNA metabolism, including transcription, ribosomal biogenesis, pre-mRNA splicing, RNA export, translation, and RNA degradation (2, 3). The RNA binding, RNA-dependent ATPase, and RNA unwinding activities of eIF4A have been studied in great detail. Analyses of the RNAactivated ATPase activity of eIF4A have demonstrated that the binding of ATP and RNA to eIF4A are coupled and that eIF4A undergoes a sequence of conformational changes as it binds substrates (RNA and ATP), hydrolyzes ATP, and releases products (4, 5). A comprehensive investigation of the unwinding activity has demonstrated that eIF4A is able to function alone as an RNA helicase and that a quantitative relationship exists between the initial rate of unwinding and the stability of the duplex (6). A simple kinetic framework for the helicase activity of eIF4A was established and indicates that duplex unwinding is non-processive (6).Recently, the helicase activity of eIF4A has been studied with respect to substrate specificity. Results confirmed that the degree of unwinding short (10 -15 base pairs) RNA duplexes by eIF4A is dependent only on the stability (rather than the length) of the duplex, and that the amplitude of unwinding may also be correlated with duplex stability (7). eIF4A was also shown to be capable of unwinding blunt-ended (no singlestranded region) RNA/DNA, 2 DNA/RNA, and RNA/DNA-PS (PS is phosphorothioa...

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.

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