Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A.

Abstract

eIF‐4A is a translation initiation factor that exhibits bidirectional RNA unwinding activity in vitro in the presence of another translation initiation factor, eIF‐4B and ATP. This activity is thought to be responsible for the melting of secondary structure in the 5′ untranslated region of eukaryotic mRNAs to facilitate ribosome binding. eIF‐4A is a member of a fast growing family of proteins termed the DEAD family. These proteins are believed to be RNA helicases, based on the demonstrated in vitro RNA helicase activity of two members (eIF‐4A and p68) and their homology in eight amino acid regions. Several related biochemical activities were attributed to eIF‐4A: (i) ATP binding, (ii) RNA‐dependent ATPase and (iii) RNA helicase. To determine the contribution of the highly conserved regions to these activities, we performed site‐directed mutagenesis. First we show that recombinant eIF‐4A, together with recombinant eIF‐4B, exhibit RNA helicase activity in vitro. Mutations in the ATPase A motif (AXXXXGKT) affect ATP binding, whereas mutations in the predicted ATPase B motif (DEAD) affect ATP hydrolysis. We report here that the DEAD region couples the ATPase with the RNA helicase activity. Furthermore, two other regions, whose functions were unknown, have also been characterized. We report that the first residue in the HRIGRXXR region is involved in ATP hydrolysis and that the SAT region is essential for RNA unwinding. Our results suggest that the highly conserved regions in the DEAD box family are critical for RNA helicase activity.

Reverse gyrase from hyperthermophiles: probable transfer of a thermoadaptation trait from Archaea to Bacteria

“…For instance, within the helicase-like domain, all reverse gyrases share the sequences AP(T/P)GXG K(T/S) (equivalent of the helicase motif I [AX 4 GKT]), DDVD (A/T) (equivalent of the DEAD helicase motif II), SAT (motif III), XRGXDXP (helicase motif V [ARGXD]), and TYXQ(A /G)SGRXSR (helicase motif VI [QXXGRXGR]). In DNA and RNA helicases, all of these motifs are assumed to form the active site: motifs I and II have been shown to be involved in the binding and hydrolysis of ATP, motif III seems responsible for the unwinding activity, and motif VI may interact with DNA or RNA in relation to ATP hydrolysis (34,35). Other motifs exclusively found in the helicase domain of reverse gyrases are the N-terminal putative zinc finger and several other amino acid blocks distributed along the sequence.…”

Multiple mammalian proteasomal ATPases, but not proteasome itself, are associated with TATA‐binding protein and a novel transcriptional activator, TIP120

“…The human proteasomal ATPases include TBP1 (Tat-binding protein 1), TBP7, S4 (subunit 4), MSS1 (mammalian suppressor of sgv1), SUG1 (suppressor of gal4), and SUG2 (Tanaka 1995;Russell et al 1996). The six proteasomal ATPases also contain conserved motifs characteristic of ATPases and RNA/DNA helicases (Gorbalenya et al 1989;Pause & Sonenberg 1992;Schmid & Linder 1992); the distance between those motifs are constant among those ATPases (Makino et al 1996).…”

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

“…The D and E residues coordinate the ATP-associated Mg 2+ and activate the attacking water molecule, respectively [109,110]. Mutation of these residues reduces ATPase and helicase reactivity [111][112][113].…”

“…The close proximity of some residues in motifs III to that in motif II suggests that motif III transduces the energy of ATP hydrolysis to the DNA [88]. Motif III mutants of UL5, UvrD, and eIF-4A exhibited uncoupling of ATPase and helicase activities [79,110,114]. The highly conserved Q in motif III contacts the -phosphate of the bound nucleotide in PcrA [89], UvrD, and UL5 [113,115].…”