Biochemical and Kinetic Characterization of the RNA Helicase Activity of Eukaryotic Initiation Factor 4A

George W Rogers,Nancy J Richter,William C Merrick

doi:10.1074/jbc.274.18.12236

George W Rogers, Nancy J Richter + Show 1 more

Open Access

https://doi.org/10.1074/jbc.274.18.12236

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Abstract

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.

Highlights

Synthesis and involves eIF3, eIF4A, eIF4B, eIF4F, and possibly eIF4H, a novel initiation factor demonstrated to interact with RNA [1, 3, 4]
That eIF4A may be able to unwind RNA duplex structures independently of other initiation factors based on the following: (i) eIF4A displays inherent ATP-dependent RNA binding and RNA-dependent ATPase activities in the absence of other initiation factors; (ii) the sequence of eIF4A contains the common motifs associated with DEAD box proteins, and in several cases it has been demonstrated that these proteins are capable of unwinding RNA duplexes in vitro (14 –17, 24); (iii) eIF4A alone has been shown to alter secondary and/or tertiary structure in reovirus mRNAs and to have a limited ability in disrupting duplexes formed by annealing short DNA oligonucleotides to globin and reovirus mRNAs [25]
Design of the Helicase Substrates—As noted in the Introduction, it was hypothesized that eIF4A may display an ATP-dependent helicase activity in the absence of accessory initiation factors

Summary

Introduction

Synthesis and involves eIF3, eIF4A, eIF4B, eIF4F, and possibly eIF4H, a novel initiation factor demonstrated to interact with RNA [1, 3, 4]. Prior studies with eIF4A have shown that an additional initiation factor, eIF4B, is a strict requirement for RNA duplex unwinding activity of eIF4A in vitro [8, 9, 22, 23] It was hypothesized, that eIF4A may be able to unwind RNA duplex structures independently of other initiation factors based on the following: (i) eIF4A displays inherent ATP-dependent RNA binding and RNA-dependent ATPase activities in the absence of other initiation factors; (ii) the sequence of eIF4A contains the common motifs associated with DEAD box (and related DEAH and DExH box) proteins, and in several cases it has been demonstrated that these proteins are capable of unwinding RNA duplexes in vitro (14 –17, 24); (iii) eIF4A alone has been shown to alter secondary and/or tertiary structure in reovirus mRNAs and to have a limited ability in disrupting duplexes formed by annealing short DNA oligonucleotides to globin and reovirus mRNAs [25]. It was reasoned that eIF4A may possess an inherent RNA duplex unwinding activity in the absence of eIF4B or other initiation factors given either increased concentrations of duplex substrate or a duplex substrate of lesser stability

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