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

Sun Y, Atas E, Lindqvist L, Sonenberg N, Pelletier J, Meller A - Nucleic Acids Res. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.

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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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.
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gks278-F1: 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.

Mentions: Before undertaking sm-FRET measurements, we verified that FRET could be used to assess eIF4AI helicase activity in bulk. As previously reported (8), the helicase activity of eIF4A was observed using a radiometric-based gel assay (Supplementary Figure S2). We then developed a bulk FRET assay for eIF4AI, using an RNA/DNA hybrid duplexes since our plan was to immobilize RNA substrates to solid support matrices using DNA for sm-FRET experiments (see below) and eIF4AI is able to unwind such substrates (30). Substrates consisting of 18-mer RNA/DNA hybrid duplexes (Tm = 51.1°C, ΔG = −21.4 kcal/mol) labeled using a FRET pair (Cy3–Cy5) located on opposite strands displayed a stable, high FRET level (Figure 1). Upon addition of 1 mM ATP and increasing concentrations of eIF4AI, we observed a gradual decrease in FRET signal, followed by a steady lower FRET state determined by eIF4AI’s concentration (Figure 1). The characteristic half-life for FRET kinetics was ∼3 min. In this assay, we used an extremely low RNA/DNA duplex concentration (1 nM) to avoid re-annealing of the two nucleic acids strands within the experimental timeframe. No FRET change was observed in the absence of ATP and the FRET signal was eIF4AI concentration dependent (Figure 1). Gel electrophoresis analysis of radio-labeled RNA duplexes showed that in the presence of ATP, eIF4AI unwinds RNA duplexes, in agreement with the FRET measurements, and consistent with previous studies (19). These experiments allow us to conclude that increasing eIF4AI concentrations lead to an increase in unwound RNA:DNA duplexes, although we can make no inferences on the linearity of the response from these experiments.Figure 1.


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

Sun Y, Atas E, Lindqvist L, Sonenberg N, Pelletier J, Meller A - Nucleic Acids Res. (2012)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401463&req=5

gks278-F1: 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.
Mentions: Before undertaking sm-FRET measurements, we verified that FRET could be used to assess eIF4AI helicase activity in bulk. As previously reported (8), the helicase activity of eIF4A was observed using a radiometric-based gel assay (Supplementary Figure S2). We then developed a bulk FRET assay for eIF4AI, using an RNA/DNA hybrid duplexes since our plan was to immobilize RNA substrates to solid support matrices using DNA for sm-FRET experiments (see below) and eIF4AI is able to unwind such substrates (30). Substrates consisting of 18-mer RNA/DNA hybrid duplexes (Tm = 51.1°C, ΔG = −21.4 kcal/mol) labeled using a FRET pair (Cy3–Cy5) located on opposite strands displayed a stable, high FRET level (Figure 1). Upon addition of 1 mM ATP and increasing concentrations of eIF4AI, we observed a gradual decrease in FRET signal, followed by a steady lower FRET state determined by eIF4AI’s concentration (Figure 1). The characteristic half-life for FRET kinetics was ∼3 min. In this assay, we used an extremely low RNA/DNA duplex concentration (1 nM) to avoid re-annealing of the two nucleic acids strands within the experimental timeframe. No FRET change was observed in the absence of ATP and the FRET signal was eIF4AI concentration dependent (Figure 1). Gel electrophoresis analysis of radio-labeled RNA duplexes showed that in the presence of ATP, eIF4AI unwinds RNA duplexes, in agreement with the FRET measurements, and consistent with previous studies (19). These experiments allow us to conclude that increasing eIF4AI concentrations lead to an increase in unwound RNA:DNA duplexes, although we can make no inferences on the linearity of the response from these experiments.Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.

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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