Mutational analysis of Has1p. (A) Boxes represent the conserved helicase motifs. The putative second Walker A motif is also shown. Positions of the mutations are indicated. (B) Cells containing wild-type HAS1 and either of the indicated has1 alleles were streaked on 5-FOA plates and incubated at 30°C. Only S228A and K389A mutants were able to support growth. (C) Strain with the S228A mutant displays a conditional phenotype, with slow growth at 16°C and no growth at 14°C. (D) Polysome analysis of the S228A mutant grown at 30°C shows a severe effect on the 40S r-subunit production and on the total level of translation. Peaks representing the ribosome subunits, mature ribosomes and polysomes are as indicated.

Aliquots (1 μg) of purified wild-type (Has1p) and mutant proteins were analyzed by SDS–PAGE, and the polypeptides were visualized by staining with Coomassie blue. The positions of marker proteins (in kDa) are indicated at the left. Proteins K92A–K389A and R376A are not shown; they were purified to the comparable purity and homogeneity.

ATP hydrolysis by the wild-type Has1p and the mutants was measured over time. Reactions were carried out at pH 6.5 with 1 mM ATP, 2 mM MgCl₂ and 390 nM proteins at 30°C in the presence of 400 ng/μl of total yeast RNA. No ATPase activity was observed without RNA (data not shown). (A) ATPase activity of Has1p (filled square) and mutant proteins K92A (filled circle), S228A (open square), T230A (open circle), H375E (open diamond) and R376A (x). (B) ATPase activity of Has1p (filled square) and mutant proteins K92A (filled circle), K389A (x) and the double mutant K92A–K389A (inverted open triangle).

ATPase stimulation of Has1p by various RNAs. (A) rRNA was recovered from the polysome fractions as shown. (B) ATPase activity was measured at pH 6.5 in the presence of 1 mM ATP, 2 mM MgCl₂ and 10 nM rRNA, tRNA, poly(A) RNA or total yeast RNA after 40 min of incubation at 30°C. Stimulation with rRNAs and poly(A) RNA was observed, with the highest values obtained with fractions 10 and 12 presumably containing 60S/pre-60S and 80S/95S complexes, respectively (). The RNA amount in the reaction was normalized to 10 μg/ml.

Unwinding of RNA/DNA duplexes by wild-type and Has1p mutants. (A) Time course for ATP-dependent unwinding of 3′ duplex and 5′ duplex by wild-type Has1p. In short, 50 nM duplex was incubated with 50 nM protein with or without 1 mM ATP, at 30°C for the time indicated in minutes. To prevent reannealing of the displaced ³²P-labeled oligonucleotide, 1 μM cold DNA oligonucleotide was added as a competitor. Products were separated on a 15% polyacrylamide gel, which was then subjected to autoradiography. (B) Unwinding activities of the wild-type and Has1p mutants. An aliquot of 50 nM duplex (3′ top panel, 5′ bottom panel) was incubated either with 50 nM protein (molar ratio 1:1, as indicated) or 10 nM protein (molar ratio 1:5, as indicated). The reactions were carried out for 60 min at 30°C in the presence of 3 mM ATP. To prevent reannealing of the displaced ³²P-labeled oligonucleotide, 1 μM cold DNA oligonucleotide was added as a competitor. Products were separated on a 15% polyacrylamide gel, which was then subjected to autoradiography. As control, the duplex was either heat-denatured for 2 min at 95°C, quick-cooled on ice and then loaded, or starting material (SM) containing the same amount of duplex was loaded. (C) Quantification of the experiments that were performed with 50 nM duplex and 50 nM enzyme, and incubated for 60 min at 30°C in the presence of 3 mM ATP. The standard errors of estimation were derived from the curve fits based on three independent sets of experiments.

ATPase activities of Has1p (filled square) and mutant proteins S228A (open square), T230A (open circle), H375E (open diamond) and K389A (x) in the presence of the increasing concentrations of total yeast (Type III) RNA (A), and RNA transcript (RNA strand of 3′ duplex used in unwinding assay) (B). Reactions were carried out for 40 min at 30°C with 1 mM ATP, 2 mM MgCl₂ and 390 nM proteins. The curves were fit to the nonlinear fit (one binding site) using GraphPad Prism 4. The standard errors of estimation were derived from the curve fits based on three independent sets of experiments.