1. Volume 11, Issue 1, Pages 127-138 (January 2003)
The Q Motif 
N.Kyle Tanner, Olivier Cordin, Josette Banroques, Monique Doère, Patrick Linder 
Molecular Cell 
Volume 11, Issue 1, Pages (January 2003)
DOI: /S (03)

2. Figure 1 Discovery of the Q Motif
(A) Alignments of the six DEAD box proteins used in the initial analysis and the consensus derived from 277 DEAD box sequences in the database. Residues shown in red are conserved at least 50% of the time. Serine and threonine are considered to be equivalent because they often replace each other in motif I. The diamonds indicate where the glycine was inserted, the arrowhead indicates the conserved glutamine (99%), and the asterisk indicates the conserved isolated phenylalanine (88%). Dashes are used to keep the sequences aligned and do not represent gaps in the alignments. The predicted secondary structures are shown below each sequence as H (helix) and E (sheets).
(B) A schematic of yeast eIF4A drawn to scale showing the positions of the conserved motifs as previously defined (Tanner and Linder, 2001). Below is an enlargement of the region containing the upstream phenylalanine, the Q motif, and the previously characterized motif I. The numbers below refer to the positions of the amino acids in eIF4A. The consensus is based on the same alignment as in (A), but the conservation of amino acids (uppercase) and functional groups (lowercase) are shown (a = F, W, Y; c = D, E, H, K, R; l = I, L, V; o = S, T; x = any amino acid). The glutamine is 17 aa upstream of motif I 88% of the time (range 14–32 aa), and the isolated aromatic group is 17 aa upstream of the Q motif 82% of the time (range 12–20 aa).
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 Q Motif Is Unique to DEAD Box Proteins
The sequence of a representative member of the helicase family in yeast is given with the derived consensus sequence shown below it for all members of that family. The consensus is based on a frequency of occurrence of at least 50%. Specific amino acids are shown in uppercase and families of similar functional groups are as indicated: − = D, E; + = H, K, R; a = F, H, W, Y; c = D, E, H, K, R; d = N, Q; l = I, L, V; • = any amino acid. The numbers show the frequency, in percentage, of the consensus group. Conservation of hydrophobic or polar functional groups is not shown to facilitate viewing.
(A) The amino-terminal sequence of yeast eIF4A as in Figure 1 but showing the conservation of amino acids between motifs.
(B) Alignment of sequences just upstream of the “core” of other helicase families found in yeast. With the exception of the DEAH family, all the families show a conserved glutamine (arrowhead) at least 95% of the time upstream of motif I. Rad3 (P06839; 42 sequences; 21 aa upstream of motif I 90% of the time; range 21–26 aa), Mtr4/Dob1 (P47047; 60 sequences; 96% 18–19 aa upstream; range 17–20 aa), Sth1 (P32597; 47 sequences; 78% 21 aa upstream; range 19–30 aa), and Dna2 (P38859; 86 sequences; 67% 17–18 aa upstream; range 16–33 aa). In contrast, DEAH helicases show a highly conserved LP, indicated with a question mark, that appears in roughly an equivalent position as the glutamine in other families (Prp2; P20095; 85 sequences; 98% 22 aa upstream; range 22–25 aa).
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 Q Motif Mutations in Yeast
Yeast cells transformed with plasmids containing the indicated mutations were grown overnight at room temperature and serially diluted in water, and 7.5 μl of each dilution was spotted onto 5-FOA-containing plates. The plates were incubated at 30°C. The numbers refer to the amount of dilution. All transformants were tested with two or more independent colonies. The plasmid control (p415) was the same for all the strains and it did not support growth. (A) Mutant variants of eIF4A transformed into the SS13-3A strain (tif1::HIS3/tif2::ADE2) were grown for 3 days. (B) Mutant variants of Ded1 transformed into the ded1::HIS3 strain, mutant variants of Fal1 transformed into the fal1::KANMX4 strain, and mutant variants of Prp5 transformed into the prp5::KANMX6 strain were grown for 4 days.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 ATPase Activity of eIF4A
(A) ATPase activity with increasing concentrations of RNA substrate. Reactions were incubated in reaction buffer with 1 mM ATP at 30°C for 75 min with 0.6 μM wild-type (•), S45A (■), and Q48E (▴), and with 1.2 μM F23A (+), F41A (O), and K71A (X). Q48A and a control reaction, containing the reaction mix but no added protein, are not shown, but they were approximately the same as K71A.
(B) Initial velocity of ATPase activity. The values shown are the mean of generally four independent reactions. Error bars are not shown for clarity, but the uncertainty associated with each curve is shown in Table 1. Reactions were incubated with 1 mM ATP at 30°C for various times with 0.2 μM protein and 0.2 μg/μl RNA. Q48A and the control (no protein) are not shown for clarity; they were about the same as K71A. Symbols are the same as in (A).
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 UV Crosslinking of 32P-ATP to eIF4A
Crosslinking reactions were done in 60-well microtiter plates using 2 μM of protein and 6.6 μCi of α-32P-ATP (300 Ci/mmole) in 10 μl of reaction buffer. The mix was kept on ice for 15 min and then irradiated in a UV Stratalinker 1800 (Stratagene), while still on ice, for the indicated times in minutes (∼1000 μjoules/min). The material was separated on a 12% Laemmli gel, Coomassie blue stained, dried, and subjected to autoradiography. Data were quantified with a Cyclone phosphorimager (Packard Instruments) and interpreted with the included OptQuant (version 3) software. The Coomassie-stained gels, showing the intact eIF4A protein, are shown below each autoradiogram. γ-32P-ATP (3000Ci/mmole) gave similar results (data not shown), indicating that hydrolysis was negligible under these conditions.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 Solved Crystal Structure of eIF4A
PDB coordinates were obtained from the authors or through Research Collaboratory for Structural Bioinformatics (RCBS; Berman et al., 2000). Coordinates were modeled on a Macintosh with SwissPDBViewer 3.7b2 (Guex and Peitsch, 1997); and the images were rendered with POV-Ray 3.1g.r2 ( Hydrogen bonds (dashed green lines) were calculated with the default settings. The Q motif is shown in salmon color and motif I is in blue.
(A) Domain 1 of eIF4A with a bound ADP from Benz et al. (1999), looking from the cleft formed between domains 1 and 2. Motifs I through III are indicated as in Tanner and Linder (2001). Not all the conserved residues of the Q motif or motif I are shown to facilitate viewing.
(B) The closed P loop (phosphate binding residues of motif I) of eIF4A, which does not show a resolved bound ligand (Johnson and McKay, 1999).
(C) The typical open P loop that is seen with other solved crystal structures of RNA and DNA helicases (Benz et al., 1999). Typically, a bound ligand (ATP, analog, phosphate, or sulfate) is resolved within the P loop pocket. The structure shown is identical to that in (A), but the nucleotide was deleted to facilitate viewing.
Molecular Cell  , DOI: ( /S (03) )