DEAD-Box Helicase Proteins Disrupt RNA Tertiary Structure Through Helix Capture

DEAD-box helicase proteins accelerate folding and rearrangements of highly structured RNAs and RNA–protein complexes (RNPs) in many essential cellular processes. Although DEAD-box proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt RNA tertiary structure. Here, we use single molecule fluorescence to show that the DEAD-box protein CYT-19 disrupts tertiary structure in a group I intron using a helix capture mechanism. CYT-19 binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then CYT-19 uses ATP to unwind the helix, liberating the product strands. Ded1, a multifunctional yeast DEAD-box protein, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on less stable misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome

Ded1 destabilizes docking of the P1 helix.

<p>(A) Representative FRET traces showing extended undocked lifetimes before redocking (left) and unwinding (right) in the presence of Ded1 and ATP (transitions shown in red). (B) Lifetime plots of the undocked states in the absence of Ded1 (black, all panels), with 50 nM (blue) or 0.2 µM (green) Ded1 and 2 mM ATP (left panel), with 0.1 µM (cyan) or 0.9 µM (orange) Ded1 and 2 mM AMP–PNP (center panel), and with 0.9 µM Ded1 and no nucleotide (red, right panel). (C) Lifetime plots of the docked state of P1 under the same conditions and represented by the same color scheme as (B). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s013" target="_blank">Data S1</a>. The calculated <i>k</i>_dock and <i>k</i>_undock values for each condition are listed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s012" target="_blank">Table S5</a>.</p

CYT-19 destabilizes tertiary docking of the P1 helix into the <i>Tetrahymena</i> ribozyme core.

<p>(A) Cartoon of the ribozyme showing P1 helix docking, undocking, and unwinding rate constants in the presence of CYT-19, with the corresponding rate constants without CYT-19 in parentheses (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s008" target="_blank">Table S1</a>). (B) Representative FRET traces and histograms showing reversible docking (transitions shown in red) without CYT-19 (top), with CYT-19 and ATP (middle traces), and with CYT-19 and AMP–PNP (bottom). (C and D) Lifetime distributions of the docked (C) and undocked (D) states without CYT-19 (black) or with 0.5 µM (blue) or 1 µM (green) CYT-19 and 2 mM ATP-Mg²⁺ (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s002" target="_blank">Figures S2</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s008" target="_blank">Tables S1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s009" target="_blank">S2</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s013" target="_blank">Data S1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s014" target="_blank">S2</a>). (E) Lifetime distributions of undocked P1 in the presence of 2 µM CYT-19 with AMP–PNP (red, left plot), without nucleotide (pink, center plot), and with ADP (orange, right plot). In each plot, corresponding data in the absence of CYT-19 and for 2 µM CYT-19 with ATP are shown for comparison in black and blue, respectively (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s008" target="_blank">Table S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s013" target="_blank">Data S1</a>).</p

CYT-19 dissociation from the ribozyme.

<p>Following a CYT-19 washout in the continued presence of 2 mM AMP–PNP, the average FRET value was followed for ribozyme molecules that started this observation period with the P1 helix undocked (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s013" target="_blank">Data S1</a>). The time evolution of the average FRET value for these molecules (red, 62 molecules) was fit by three phases with rate constants and relative amplitudes of 30 min⁻¹ (0.36), 0.43 min⁻¹ (0.29), and 0.01 min⁻¹ (0.35). We infer that the rate constant of 0.43 min⁻¹ reflects CYT-19 dissociation because this phase was not observed in the absence of CYT-19. The initial fast phase reflects P1 docking re-equilibration with bound CYT-19 and is predicted from the model, and the slowest phase most likely reflects the slow conversion of ribozyme molecules that initially give poor docking or are misfolded (see Results, “CYT-19 Can Remain Associated with the Ribozyme for Multiple Cycles of Helix Capture”). In the absence of CYT-19 (black, 64 molecules), re-equilibration of P1 docking gave a single observed phase of 130 min⁻¹ (inset). The endpoint is lower (0.73) than expected (0.85, indicated by dashed line), most likely reflecting molecules that dock P1 poorly as above.</p

CYT-19–mediated unwinding of a shorter P1 helix (6 bp) is rate limited by spontaneous loss of tertiary contacts.

<p>(A) Cartoon representation showing docking, undocking, and unwinding rate constants for the 6-bp P1 helix in the presence of CYT-19. Rate constants in the absence of CYT-19 are shown in parentheses and are similar to previous values <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981-Bartley1" target="_blank">[25]</a>. (B) Representative FRET traces and histograms (transitions shown in red) in the absence of CYT-19 (top) and with 1 µM CYT-19 and AMP–PNP (bottom). (C) Lifetime distributions of the docked (top) and undocked (bottom) states in the absence of CYT-19 (black, 102 molecules; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s013" target="_blank">Data S1</a>) and with 1 µM CYT-19 and AMP–PNP (blue, 163 molecules; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s013" target="_blank">Data S1</a>). (D) CYT-19 unwinding of the P1 helix monitored by ensemble techniques. The maximum observed unwinding rate constant (<i>k</i>_max) for the standard 6-bp P1 helix is 6 min⁻¹ (red). Weakening P1 docking by atomic mutagenesis (blue, −3 m, rSA₅) increases <i>k</i>_max to 20 min⁻¹, and strengthening the docking contacts (green, rP, also in inset) decreases <i>k</i>_max to 0.075 min⁻¹ (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s006" target="_blank">Figure S6</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981.s010" target="_blank">Table S3</a>). Error bars represent the standard deviation of at least two independent measurements.</p

Model for RNA tertiary structure disruption by helix capture.

<p>DEAD-box proteins (orange) associate with structured RNAs nonspecifically (left), which can result in the helicase core being positioned to interact with transiently exposed helices (center). This interaction prevents reformation of tertiary contacts by the bound helix, destabilizing the RNA tertiary structure and allowing DEAD-box proteins to use ATP to perform helix unwinding (right). The DEAD-box protein illustrated is the yeast ortholog of CYT-19, Mss116 (pdb 3I5X), and the <i>Tetrahymena</i> ribozyme shown is a model structure presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001981#pbio.1001981-Lehnert1" target="_blank">[62]</a>.</p