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A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation.

Lu J, Jiang C, Li X, Jiang L, Li Z, Schneider-Poetsch T, Liu J, Yu K, Liu JO, Jiang H, Luo C, Dang Y - Nucleic Acids Res. (2015)

Bottom Line: Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation.Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI.Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Metabolism and Molecular Medicine, the Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

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Functions of additional linker residues in regulating the activity of eIF4AI. (A) Linker residues that face solvent and do not take part in the hydrophobic core. (B) ATPase activity of eIF4AI mutants under saturating RNA concentration (250 μg/ml). The enzyme concentration was 0.4 μM. (C) RNA helicase activity of eIF4AI mutants under saturating ATP concentration (2 mM). (D) In vitro translation efficiencies of WT and mutant eIF4AI. The error bar represents SEM (n = 3).
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Figure 6: Functions of additional linker residues in regulating the activity of eIF4AI. (A) Linker residues that face solvent and do not take part in the hydrophobic core. (B) ATPase activity of eIF4AI mutants under saturating RNA concentration (250 μg/ml). The enzyme concentration was 0.4 μM. (C) RNA helicase activity of eIF4AI mutants under saturating ATP concentration (2 mM). (D) In vitro translation efficiencies of WT and mutant eIF4AI. The error bar represents SEM (n = 3).

Mentions: The aforementioned simulation and experimental results suggest an important role of the inter-domain linker in the regulation of eIF4AI's activity. However, compared with other conserved motifs, this linker has attracted less attention. A previous study reported mutations of two polar residues in the linker – K237 and E240, which are located next to V236 and L241, respectively (Figure 6A), also increased eIF4AI's ATPase activity. But the unwinding activities of those mutants were not determined (13). We then performed mutational studies on these two residues as well to characterize the role of the linker in coupling the ATPase to the helicase activity. In addition, we mutated another hydrophobic residue on the linker, L243, which does not take part in forming the hydrophobic core. Unexpectedly, although E240A and L243G increased ATPase activities as other linker mutations did (Figure 6B, Table 2 and Supplementary Figure S7E), the unwinding activity of eIF4AI was also increased by those two mutations (Figure 6C and Supplementary Figure S7F). On the other hand, eIF4AI mutant with K237A showed a similar helicase activity compared with wild type (Figure 6C and Supplementary Figure S7F).


A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation.

Lu J, Jiang C, Li X, Jiang L, Li Z, Schneider-Poetsch T, Liu J, Yu K, Liu JO, Jiang H, Luo C, Dang Y - Nucleic Acids Res. (2015)

Functions of additional linker residues in regulating the activity of eIF4AI. (A) Linker residues that face solvent and do not take part in the hydrophobic core. (B) ATPase activity of eIF4AI mutants under saturating RNA concentration (250 μg/ml). The enzyme concentration was 0.4 μM. (C) RNA helicase activity of eIF4AI mutants under saturating ATP concentration (2 mM). (D) In vitro translation efficiencies of WT and mutant eIF4AI. The error bar represents SEM (n = 3).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4666354&req=5

Figure 6: Functions of additional linker residues in regulating the activity of eIF4AI. (A) Linker residues that face solvent and do not take part in the hydrophobic core. (B) ATPase activity of eIF4AI mutants under saturating RNA concentration (250 μg/ml). The enzyme concentration was 0.4 μM. (C) RNA helicase activity of eIF4AI mutants under saturating ATP concentration (2 mM). (D) In vitro translation efficiencies of WT and mutant eIF4AI. The error bar represents SEM (n = 3).
Mentions: The aforementioned simulation and experimental results suggest an important role of the inter-domain linker in the regulation of eIF4AI's activity. However, compared with other conserved motifs, this linker has attracted less attention. A previous study reported mutations of two polar residues in the linker – K237 and E240, which are located next to V236 and L241, respectively (Figure 6A), also increased eIF4AI's ATPase activity. But the unwinding activities of those mutants were not determined (13). We then performed mutational studies on these two residues as well to characterize the role of the linker in coupling the ATPase to the helicase activity. In addition, we mutated another hydrophobic residue on the linker, L243, which does not take part in forming the hydrophobic core. Unexpectedly, although E240A and L243G increased ATPase activities as other linker mutations did (Figure 6B, Table 2 and Supplementary Figure S7E), the unwinding activity of eIF4AI was also increased by those two mutations (Figure 6C and Supplementary Figure S7F). On the other hand, eIF4AI mutant with K237A showed a similar helicase activity compared with wild type (Figure 6C and Supplementary Figure S7F).

Bottom Line: Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation.Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI.Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Metabolism and Molecular Medicine, the Ministry of Education, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

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Related in: MedlinePlus