Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Oct 23;425(20):3839-45.
doi: 10.1016/j.jmb.2013.05.006. Epub 2013 May 20.

AMP Sensing by DEAD-box RNA Helicases

Affiliations
Free PMC article

AMP Sensing by DEAD-box RNA Helicases

Andrea A Putnam et al. J Mol Biol. .
Free PMC article

Abstract

In eukaryotes, cellular levels of adenosine monophosphate (AMP) signal the metabolic state of the cell. AMP concentrations increase significantly upon metabolic stress, such as glucose deprivation in yeast. Here, we show that several DEAD-box RNA helicases are sensitive to AMP, which is not produced during ATP hydrolysis by these enzymes. We find that AMP potently inhibits RNA binding and unwinding by the yeast DEAD-box helicases Ded1p, Mss116p, and eIF4A. However, the yeast DEAD-box helicases Sub2p and Dbp5p are not inhibited by AMP. Our observations identify a subset of DEAD-box helicases as enzymes with the capacity to directly link changes in AMP concentrations to RNA metabolism.

Keywords: AMP; ATPase; DEAD-box; RNA helicase; unwinding.

Figures

Figure 1
Figure 1. Nucleotide to Ded1p
Binding of ATP (red), ADP (blue), and AMP (green) to Ded1p in the absence of RNA.Black circles show a control reaction (ADP titration) without Ded1p. Data points represent averages from at least four independent measurements, error bars mark one standard deviation. Solid lines show the best fit to the binding isotherm. Obtained data are compiled in Supplementary Table S1. Nucleotide binding was monitored by competition with mantADP (Supplementary Fig.S2). Steady-state fluorescence was measured in a buffer containing 40 mM Tris·HCl (pH 8.0), 50 mM NaCl, 0.5 mM MgCl2, 2 mM DTT, 0.01% (vol/vol) IGEPAL, and 1 μM Ded1p at 19°C,using a FluoroMax-3 spectrofluorimeter (JobinYvon, Spex Instruments S.A., Inc.) and a 3×3×35 mm quartz cuvette with 150 μl of reaction buffer. Purified Ded1p was expressed and purified as previously described . Tryptophan residues were excited at 295 nm (2 nm bandwidth) to minimize inner filter effects of the nucleotide in solution. Emission was monitored from 305-500 nm (2 nm bandwidth). MantADP (mADP) was excited at 360 nm (1 nm bandwidth) and emission was monitored from 400-500 nm (1nm bandwidth). All reactions contained equimolar concentrations of nucleotide and Mg2+ and 0.5 mM Mg2+ excess over the nucleotide. Affinities of unlabeled ATP, ADP and AMP were measured by competitive binding with 50 μM mADP.
Figure 1
Figure 1. Nucleotide to Ded1p
Binding of ATP (red), ADP (blue), and AMP (green) to Ded1p in the absence of RNA.Black circles show a control reaction (ADP titration) without Ded1p. Data points represent averages from at least four independent measurements, error bars mark one standard deviation. Solid lines show the best fit to the binding isotherm. Obtained data are compiled in Supplementary Table S1. Nucleotide binding was monitored by competition with mantADP (Supplementary Fig.S2). Steady-state fluorescence was measured in a buffer containing 40 mM Tris·HCl (pH 8.0), 50 mM NaCl, 0.5 mM MgCl2, 2 mM DTT, 0.01% (vol/vol) IGEPAL, and 1 μM Ded1p at 19°C,using a FluoroMax-3 spectrofluorimeter (JobinYvon, Spex Instruments S.A., Inc.) and a 3×3×35 mm quartz cuvette with 150 μl of reaction buffer. Purified Ded1p was expressed and purified as previously described . Tryptophan residues were excited at 295 nm (2 nm bandwidth) to minimize inner filter effects of the nucleotide in solution. Emission was monitored from 305-500 nm (2 nm bandwidth). MantADP (mADP) was excited at 360 nm (1 nm bandwidth) and emission was monitored from 400-500 nm (1nm bandwidth). All reactions contained equimolar concentrations of nucleotide and Mg2+ and 0.5 mM Mg2+ excess over the nucleotide. Affinities of unlabeled ATP, ADP and AMP were measured by competitive binding with 50 μM mADP.
Figure 2
Figure 2. Thermodynamic framework for nucleotide and RNA binding by Ded1p
(a) Apparent equilibrium dissociation constants (K1/2) for the minimal thermodynamic scheme of nucleotide and RNA binding to Ded1p(E: Enzyme, R: RNA). Constants were determined by global fitting the fraction of inorganic phosphate generated during ATP hydrolysis assays (for experimental details and data analysis see Supplementary Methods). (b) Quality assessment of the global data fit. To visualize the goodness of fit to the data set, experimental data (y-axis) were plotted versus the calculated f[Pi] for the entire model (x-axis). Data points for ADP are blue, those for AMP green. Black lines represent a linear fit through the data. The correlation coefficient is given as R2. For quality assessment of data subsets (e.g., ATP alone, high and low RNA or ADP/AMP) see Supplementary Fig.S4b-f. (c) Binding of a 10 nt single stranded RNA to 1 μM Ded1p without nucleotide (black), and with saturating ATP (red), ADP (blue), or with AMP (green). All reactions contained equimolar concentrations of nucleotide and Mg2+ and 0.5 mM Mg2+ excess over the nucleotide. For RNA sequences see Supplementary Materials. Fractions of bound RNA with ADP, AMP and without nucleotide were determined from the equilibrium constants shown in panel (a). Nucleotide concentrations were extrapolated to infinity. Error bars represent the 95% confidence interval obtained from the global fit of all data to the complete thermodynamic model (Supplementary Methods, Supplementary Table S1). (d) Cooperativity between RNA and nucleotide binding, as calculated from the difference in free energies for nucleotide binding with and without RNA. (ΔΔG° = ΔG° (nt)-RNA − ΔG° (nt)+RNA, with ΔG° = −RTln(1/K1/2)). Positive ΔΔG° values indicate cooperativity between nucleotide and RNA binding, negative values indicate anti-cooperative binding.
Figure 3
Figure 3. Impact of AMP on RNA helicase activity
(a) Effect of adenosine nucleotides on duplex unwinding by Ded1p. Representative non-denaturing PAGE of unwinding time courses (30 min) with Ded1p.Cartoons show the mobility of unwound and duplex substrates, the asterisk marks the radiolabel. Unwinding reactions for Ded1p were performed as previously described (19 °C, 20 μl reaction buffer containing 40 mMTris·HCl (pH 8.0), 50 mMNaCl, 0.5 mM MgCl2, 2 mM DTT, 0.6 unit•μl−1RNasin, 0.01% (vol/vol) IGEPAL, 0.1 μM Ded1p, 0.5 nM 13 bp RNA duplex). Ded1p was pre-incubated with the 13 bp duplex substrate with a 25 nt 3’ss overhang (for sequences see Supplementary Materials) and the reactions were initiated with 0.5 mM ATP in the presence or absence of 0.5 mMindicated nucleotide (Total [Mg2+]: 1mM with ATP alone, 1.5 mM in all other reactions). At each time-point, a 3 μl portion was removed, and the reaction was stopped with 3 μl of 1% SDS, 50 mM EDTA, 20% (v/v) Glycerol, and 0.1% (w/v) Bromphenol Blue/Xylene Cylanol. Samples were applied to a 15% native polyacrylamide gel, and electrophoresed at 20 V/cm at 4°C. Gels were dried and radioactivity was quantified with a PhosphorImager (GE) and the ImageQuant software (Mol. Dynamics).First order unwinding rate constants were calculated as previously described .(b) Observed first order unwinding rate constants (kunw) in the presence of indicated nucleotides, calculated for Ded1p from panel (a) and for several DEAD-box helicases (Mss116p, eIF4A, Sub2p, Dbp5p) and the non-DEAD-box RNA helicase Mtr4p. Error bars mark one standard deviation of at least three independent experiments. Unwinding reactions with the other RNA helicases were measured as described above, with the following modifications to obtain optimal unwinding rate constants: Mss116p: 10 mM Tris·HCl (pH 7.5), 100 mM KCl, Sub2p: 10 mM Tris·HCl (pH 7.0), 50 mM KCl at 30°C, Dbp5p: 40 mM Hepes (pH 7.5) at 30°C, eIF4A: 40 mM Hepes (pH 7.5), 8% (vol/vol) PEG6000 at 25°C and a 10bp duplex, and Mtr4p: 40 mM MOPS (pH 6.5), 100 mM NaCl, (16 bp duplex) at 30°C. Mtr4p was expressed and purified as described ,. eIF4A was expressed and purified according to a protocol identical to that described for Ded1p . Mss116p, Dbp5p, and Sub2p were generous gifts from Dr. Alan Lambowitz (University of Texas, Austin, TX), Dr. Karsten Weis (University of California, Berkeley, CA), and Dr. DitlevBroderson (University of Aarhus, Aarhus, Denmark). The proteins were expressed and purified as described ,. Radiolabeled substrates were prepared as previously described .
Figure 4
Figure 4. Modulation of the helicase activity of Ded1p to stress-induced changes in nucleotide concentrations
Upper panel: Simulated changes in nucleotide levels in response to metabolic stress, such as glucose depletion. Curves were calculated as described by Hardie et al., based on published parameters for the adenylate kinase reaction . For the calculation, the reaction is assumed at equilibrium with a constant total concentration of adenylate nucleotides. Experimental measurements of AMP concentrations suggest that this simulation might underestimate the AMP increase associated with metabolic stress. Lower panel: Response of Ded1p (represented as ATP bound state of the enzyme) to ATP depletion (red), inhibition by ADP (blue), or inhibition by AMP (green), using affinities given in Fig. 2a.

Similar articles

See all similar articles

Cited by 13 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback