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. 2013 Dec 3;21(12):2197-207.
doi: 10.1016/j.str.2013.09.016. Epub 2013 Oct 31.

Dynamic recognition of the mRNA cap by Saccharomyces cerevisiae eIF4E

Seán E O'Leary  1 , Alexey Petrov, Jin Chen, Joseph D Puglisi
Affiliations

Dynamic recognition of the mRNA cap by Saccharomyces cerevisiae eIF4E

Seán E O'Leary et al. Structure. .

Abstract

Recognition of the mRNA 5' m⁷G(5')ppp(5')N cap is key to translation initiation for most eukaryotic mRNAs. The cap is bound by the eIF4F complex, consisting of a cap-binding protein (eIF4E), a "scaffold" protein (eIF4G), and an RNA helicase (eIF4A). As a central early step in initiation, regulation of eIF4F is crucial for cellular viability. Although the structure and function of eIF4E have been defined, a dynamic mechanistic picture of its activity at the molecular level in the eIF4F·mRNA complex is still unavailable. Here, using single-molecule fluorescence, we measured the effects of Saccharomyces cerevisiae eIF4F factors, mRNA secondary structure, and the poly(A)-binding protein Pab1p on eIF4E-mRNA binding dynamics. Our data provide an integrated picture of how eIF4G and mRNA structure modulate eIF4E-mRNA interaction, and uncover an eIF4G- and poly(A)-independent activity of poly(A)-binding protein that prolongs the eIF4E·mRNA complex lifetime.

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Figures

Figure 1
Figure 1
A single-molecule FRET assay for eIF4E activity. (A) Schematic of the Cy5-eIF4E•eIF4G-4EBD complex based on the solution structure in the presence of m7GDP (Gross et al., 2003; PDB 1RF8). The schematic indicates the location of the Cy5 label attached to residue 124. (B) Experimental design for single-molecule FRET, showing immobilization of the capped RNA by annealing to a 5′-Cy3-labeled, 3′-biotinylated DNA oligonucleotide. (C) Representative single-molecule FRET trace showing FRET events due to binding of Cy5-eIF4E to the immobilized, Cy3-labeled RNA. (D) Representative arrival time distribution for binding of Cy5-eIF4E to the Cy3-RNA, with single-exponential fit. (E) Dependence of the rate of formation of the Cy5-eIF4E•cap-RNA complex on the eIF4E concentration, with linear fit. (F) Dependence of dissociation rate of the Cy5-eIF4E•cap-RNA complex on the Cy5-eIF4E concentration. (G) FRET distribution for the Cy5-eIF4E•cap-RNA complex, fit to a single Gaussian function. The number of single-molecule traces (n) used to construct distributions is given where appropriate. Error bars in kinetic plots represent standard errors for three separate measurements. See also Figure S1.
Figure 2
Figure 2
Modulation of eIF4E•cap-poly(CU) binding kinetics by the eIF4G 4E-binding domain and by eIF4G1. (A) Schematic of experimental design. (B) Domain architecture of S. cerevisiae eIF4G1, showing relative positions of RNA-, eIF4E-, Pab1p-, and eIF4A-binding domains, and the region of the protein constituting eIF4E-4EBD. (C) Dependence of Cy5-eIF4E-RNA association rate on eIF4G-4EBD concentration, measured at 7.5 nM Cy5-eIF4E. (D) Dependence of observed rate of dissociation of the Cy5-eIF4E•RNA complex on the concentration of eIF4G-4EBD. (E) Representative FRET distribution for the Cy5-eIF4E•RNA complex (in the presence of 125 nM eIF4G-4EBD), fit to a single Gaussian function. (F) Dependence of Cy5-eIF4E-RNA association rate on full-length eIF4G1 concentration, measured at 7.5 nM Cy5-eIF4E. (G) Dependence of observed rate of dissociation of the Cy5-eIF4E•RNA complex on the concentration of eIF4G1. (H) Representative FRET distribution for the Cy5-eIF4E•RNA complex (in the presence of 125 nM eIF4G1), fit to the sum (grey line) of two Gaussian functions (red lines). The number of single-molecule traces (n) used to construct distributions is given where appropriate. Error bars in kinetic plots represent standard errors for single-exponential fits. See also Figure S2.
Figure 3
Figure 3
Effects of RNA secondary structure on kinetics of binding of Cy5-eIF4E to immobilized RNA. (A) Schematic of experimental design. (B) Sequences of structured RNAs and hybrid duplex with labeled DNA oligonucleotides, indicating positions of hairpins, Cy3 label, and biotin. (C) Rates of association of Cy5-eIF4E (7.5 nM) with immobilized poly(CU) and poly(CU)10hp12 RNAs, and with poly(CU)10hp12 RNA in the presence of eIF4G-4EBD (150 nM). (D) Rates of dissociation of the Cy5-eIF4E•poly(CU), Cy5-eIF4E•poly(CU)10hp12, and Cy5-eIF4E•4EBD•poly(CU)10hp12 complexes. (E) Apparent dissociation constants for the Cy5-eIF4E•poly(CU), Cy5-eIF4E•poly(CU)10hp12, and Cy5-eIF4E•4EBD•poly(CU)10hp12 complexes, computed from the quotient koff/kon. Error bars in kinetic plots represent standard errors for single-exponential fits. See also Figure S3.
Figure 4
Figure 4
A long-lived, high-FRET state for the Cy5-eIF4E•RNA complex observed in the presence of Pab1p. (A) Schematic of experimental design. The domain architecture of eIF4G1 is abridged from the full disposition of domains (Figure 2B) by combining the three separate RNA binding domains and omitting the eIF4A-binding domain. Pab1p-bd refers to the eIF4G1 Pab1p-binding domain. (B) Representative single-molecule FRET trace showing binding of Cy5-eIF4E to immobilized, cap-poly(CU) RNA in the presence of 2 μM Pab1p. (C) Dependence of Cy5-eIF4E-mRNA association rate on factor composition. (D) Dependence of dissociation rate of the Cy5-eIF4E•mRNA complex in the presence of eIF4G1 (250 nM), Pab1p (2 μM), and eIF4A (400 nM). (E) – (H) FRET intensity distributions for the Cy5-eIF4E complexes in (D). The number of single-molecule traces (n) used to construct distributions is given where appropriate. Error bars in kinetic plots represent standard errors for single-exponential fits. See also Figure S4.
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