The Q motif and flanking sequences. (A) The amino-acid (aa) sequence of Ded1 around the Q motif (residues 142–193). The predicted helical (H) structure is shown below (PredictProtein; Rost and Sander, 1994), as well as the consensus from 277 DEAD-box sequences in the database (a=F,Y,W; c=D,E,H,K,R; −=D,E; +=H,K,R; l=I,L,V; ○=S, T; h=A,I,L,M,P,V; •=any amino acid; Bork Consensus; http://www.bork.embl-heidelberg.de). The numbers indicate the percentage of occurrence of each residue or functional group. (B) Predicted network of interactions of Ded1 of the Q motif and flanking sequences based on the solved crystal structure of eIF4A with a bound ADP (Benz et al, 1999). The sidechain (s) of the highly conserved Q169 interacts with the N6 and N7 positions of adenine and with the peptide backbone (b) of motif I. The conserved aromatic group (F162) forms hydrophobic stacking interactions (h) with the base. ^*, A highly conserved, but isolated, aromatic group (F and less often W) that typically appears 17 aa upstream of the Q motif; , the essentially invariant Gln that typically appears 17 aa upstream of motif I. Note that a short helix is invariably predicted to occur between the isolated upstream aromatic residue and the Q motif within randomly selected sequences in the database.

Complementation of the Q motif mutants in yeast. (A) Representative growth of yeast transformed with various mutants at different temperatures. Cells were grown in liquid medium lacking leucine to approximately the same density, serially diluted, and each dilution was spotted onto 5-FOA-containing plates. Plates were incubated at the indicated temperatures for 3 days (30 and 36°C) or 4 days (18°C). WT, wild-type Ded1; p415, plasmid alone control. (B) Resume of the in vivo analysis. Residues in bold and underlined were made by site-specific mutagenesis; all others were made with oligonucleotides that were completely randomized at the three nucleotide positions of the codon. Proteins of the boxed residues were purified. WT, wild-type or nearly wild-type growth; TS/CS, temperature-sensitive or cold-sensitive phenotypes; NG, no detectable growth.

ATPase activities of the wild type and selected mutants. (A) ATPase activity using increasing concentrations of whole yeast (type III) RNA. All comparisons were made with the same lot of RNA. Reactions were carried out for 40 min at 30°C with 1 mM ATP and 15 nM proteins. (B) Michaelis–Menten kinetics of the ATPase reaction using 15 nM of protein and 400 ng/μl of whole yeast RNA. The standard deviations, from three independent sets of experiments, were deleted from some points because they were not significant. Reaction velocities were determined from the initial linear range of the reaction, which corresponded to less than 7% reaction. The reaction profiles of F162A, Q169A and Q169E are not shown; they were slightly above K192A at this plot scale. (C) Competitive inhibition of the ATPase reaction by ADP. ○, wild type; ×, T166A; +, T166S; ⋄, F162L; □, K192A.

Electrophoretic mobility shift of Ded1-bound 44-mer RNA. In all, 2.5 nM of ³²P-labeled RNA (shown in Figure 5) was incubated with the indicated concentrations of Ded1 (in μM) and electrophoretically separated on a 7.5% nondenaturing polyacrylamide gel that was run at 4°C. The ADP and AMP-PNP (PNP) concentrations were 5 mM. We could account for most of the added radioactivity by quantifying the gels with a phosphoimager. There was little or no RNA degradation, and the apparent loss of radioactivity in some lanes is caused by smearing, which is more obvious on longer exposures. ^*, free RNA; −, origin of gel.

Strand-displacement activity of Q motif mutants. (A) RNA–DNA duplex used for the unwinding assay. The DNA oligonucleotide was 5′-³²P-labeled and hybridized to unlabeled RNA. The calculated ΔG° of the duplex was −19.8 kcal/mol, which gave a T_m of ∼52°C under our salt and duplex concentrations. (B) Time course for ATP-dependent unwinding of the duplex. In all, 200 nM of duplex was incubated with 30 nM (wild type) or 120 nM (mutant) protein with 1 mM ATP, for the indicated times in minutes at 37°C. To prevent reannealing of the displaced ³²P-labeled oligonucleotide, 5 μM of cold DNA oligonucleotide was added as a competitor. Products were separated on a 15% polyacrylamide gel, which was then subjected to autoradiography. ∣, free DNA oligonucleotide; ∥, RNA–DNA duplex; HD, heat-denatured at 95°C and then quick-cooled on ice. (C) Relative helicase activity of mutants compared to the wild type, based on the extent of reaction after 20 min at various protein concentrations. All values were normalized to the same protein concentration. Standard errors are as shown.