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. 2012 Jul;40(13):6199-207.
doi: 10.1093/nar/gks278. Epub 2012 Mar 28.

The eukaryotic initiation factor eIF4H facilitates loop-binding, repetitive RNA unwinding by the eIF4A DEAD-box helicase

Yingjie Sun  1 , Evrim Atas, Lisa Lindqvist, Nahum Sonenberg, Jerry Pelletier, Amit Meller
Affiliations

The eukaryotic initiation factor eIF4H facilitates loop-binding, repetitive RNA unwinding by the eIF4A DEAD-box helicase

Yingjie Sun et al. Nucleic Acids Res. 2012 Jul.

Abstract

Eukaryotic translation initiation is a highly regulated process in protein synthesis. The principal translation initiation factor eIF4AI displays helicase activity, unwinding secondary structures in the mRNAs 5'-UTR. Single molecule fluorescence resonance energy transfer (sm-FRET) is applied here to directly observe and quantify the helicase activity of eIF4AI in the presence of the ancillary RNA-binding factor eIF4H. Results show that eIF4H can significantly enhance the helicase activity of eIF4AI by strongly binding both to loop structures within the RNA transcript as well as to eIF4AI. In the presence of ATP, the eIF4AI/eIF4H complex exhibits persistent rapid and repetitive cycles of unwinding and re-annealing. ATP titration assays suggest that this process consumes a single ATP molecule per cycle. In contrast, helicase unwinding activity does not occur in the presence of the non-hydrolysable analog ATP-γS. Based on our sm-FRET results, we propose an unwinding mechanism where eIF4AI/eIF4H can bind directly to loop structures to destabilize duplexes. Since eIF4AI is the prototypical example of a DEA(D/H)-box RNA helicase, it is highly likely that this unwinding mechanism is applicable to a myriad of DEAD-box helicases employed in RNA metabolism.

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Figures

Figure 1.
Figure 1.
Bulk FRET assay displays an eIF4AI-dependent decrease in the steady-state FRET level. (A) A schematic diagram showing the substrate molecules for bulk FRET assay. An 18 base pair RNA/DNA duplex labeled with a FRET pair introduced at a very low concentration (1 nM) is unwound only in the presence of ATP and eIF4AI, reaching a lower steady-state FRET level. (B) Results from bulk FRET assays monitoring eIF4AI helicase activity at the indicated eIF4AI concentrations and 1 mM ATP.
Figure 2.
Figure 2.
sm-FRET recordings of hairpin substrates in the presence of eIF4AI. (A) Schematic diagram illustrating DNA/RNA hairpin substrate harboring a 12-nt loop and 12 nt duplex region, labeled with a FRET pair (Cy3–Cy5). The substrate is immobilized to a glass surface via the 3′-end biotin. (B) sm-FRET of the immobilized RNA/DNA hairpin in the presence of eIF4AI, displaying episodes consisting of decreased FRET signals followed by return to original values. (C) FRET distribution from individual sm traces (N = 38 out of 1000) showing two distinct peaks at 0.9 and 0.6.
Figure 3.
Figure 3.
sm-FRET recordings of hairpin substrates in the presence of eIF4AI/eIF4H. (A) A representative sm time trace of an RNA/DNA hairpin structure (Schematic Illustration in Figure 2A). The hairpin molecule is immobilized to the cover-slip surface using a biotin moiety. eIF4AI/eIF4H are introduced at a concentration of 200 nM and ATP concentration is 0.1 mM. Donor (formula image) and acceptor (formula image) intensities (green and red, respectively) are measured and the corresponding FRET trace is calculated by: formula image (blue). (B) Histogram of FRET levels obtained from N > 2000 individual molecules under the same conditions as in A, showing two prominent peaks at 0.9 and 0.4. The oscillating FRET behavior is observed in over 50% of the sm events and is ATP dependent. (C) Dwell times of the low- and high-FRET states are used to construct the time histograms of the unwinding time and waiting time intervals (blue and green, respectively). Single exponential models are used to fit the data (black lines), yielding the characteristic timescales formula image and formula image.
Figure 4.
Figure 4.
eIF4AI/eIF4H complex remains bound to the hairpin substrate during cycles of repetitive FRET signal oscillations. A typical FRET trace for a single substrate molecule acquired in reactions containing: (A) both eIF4AI, eIF4H (1 µM) and ATP (1 mM); (B) after>5 × volume flushing of the chamber with buffer lacking eIF4AI, eIF4H and ATP (‘Wash 1’); (C) following reintroduction of only ATP at 1 mM (‘Wash 2’).
Figure 5.
Figure 5.
eIF4AI/eIF4H complex requires hairpin substrates containing minimum loop sizes of 6 nt for repetitive unwinding. The fraction of sm traces showing > 3 oscillations in a 60 s monitoring time window is plotted as a function of loop length (in nucleotides). Each data point consists of at least 1500 sm traces.
Figure 6.
Figure 6.
Dependence of the hairpin substrate unwinding dynamics by the eIF4AI/eIF4H complex on ATP or ATP-γS concentrations. In these experiments, 12-nt hairpin loop structures were used, and 1 µM of both eIF4AI and eIF4H. (A) Relationship of the waiting times (formula image, circles) and the unwinding times (formula image, triangles) on the ATP concentration. Each data point represents statistics of at least 1000 sm traces (detailed distributions are provided in the Supporting Information file). (B) The dependence of waiting times (formula image, circles) and unwinding times (formula image, triangles) on the relative ATP-γS concentration, with a fixed ATP concentration of 0.1 mM. The abscissa formula image. Each data point represents statistics of at least 500 sm traces.
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