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. 2015 Jun 26;348(6242):1486-8.
doi: 10.1126/science.aaa5089.

RNA BIOCHEMISTRY. Factor-dependent processivity in human eIF4A DEAD-box helicase

Cuauhtémoc García-García  1 , Kirsten L Frieda  2 , Kateryna Feoktistova  3 , Christopher S Fraser  3 , Steven M Block  4
Affiliations

RNA BIOCHEMISTRY. Factor-dependent processivity in human eIF4A DEAD-box helicase

Cuauhtémoc García-García et al. Science. .

Abstract

During eukaryotic translation initiation, the small ribosomal subunit, assisted by initiation factors, locates the messenger RNA start codon by scanning from the 5' cap. This process is powered by the eukaryotic initiation factor 4A (eIF4A), a DEAD-box helicase. eIF4A has been thought to unwind structures formed in the untranslated 5' region via a nonprocessive mechanism. Using a single-molecule assay, we found that eIF4A functions instead as an adenosine triphosphate-dependent processive helicase when complexed with two accessory proteins, eIF4G and eIF4B. Translocation occurred in discrete steps of 11 ± 2 base pairs, irrespective of the accessory factor combination. Our findings support a memory-less stepwise mechanism for translation initiation and suggest that similar factor-dependent processivity may be shared by other members of the DEAD-box helicase family.

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Figures

Fig. 1
Fig. 1. Experimental geometry of the single-molecule eIF4A helicase assay (not to scale)
(A) A single 72-bp RNA reporter hairpin (red) is tethered between two microscopic avidincoated beads (blue) held in optical traps (pink) by a DNA handle and a biotinylated RNA polymerase (green) transcriptionally stalled at a biotin-avidin roadblock (yellow). The tether contains a short single-stranded RNA flanking sequence adjacent to the 5′ side of the hairpin for loading eIF4A helicase, shown here complexed with eIF4B and eIF4G. (B) As eIF4A, alone or bound to combinations of eIF4B, eIF4H, and eIF4G, translocates along the RNA, its helicase activity unwinds the hairpin, leading to an increase in distance (Dx) between the trapped beads. In all figures, “eIF4G” corresponds to the truncation mutant eIF4G682-1105.
Fig. 2
Fig. 2. Factordependent processivity of eIF4A
(A) Representative singlemolecule records of helicase activity of eIF4A (purple), eIF4A•B (yellow), eIF4A•H (black), eIF4A•G (green), eIF4A•B•G (red), and eIF4A•H•G (blue) over a 60-s interval under constant load; traces are offset vertically for clarity. Note instances of forward motion corresponding to hairpin unwinding (extension increase) and rearward motion corresponding to hairpin reannealing (extension decrease). The final extension increase leading to full opening of the hairpin was facilitated by force, once the ever-shortening duplex region remaining became unstable under the constant load (black arrows; red and blue traces). (B) Normalized power spectra of the pairwise distances derived from multiple records of helicase and cofactor activities, color-coded as in (A), showing prominent peaks at a spatial frequency (0.09 bp−1, gray line) corresponding to an ~11-bp step. Each power spectrum represents an average of at least 50 different single-molecule records.
Fig. 3
Fig. 3. Translocation properties of eIF4A helicase and accessory factor combinations
(A) Forward-to-backward stepping ratio for the indicated complexes (legend, bottom), with errors (SEM). (B) Mean pause lifetime with errors (SEM) for the indicated complexes (legend, bottom). (C) eIF4A•B•G melting of a reporter hairpin with the sequence shown (left), color-coded according to the legend (bottom). Five representative single-molecule records, expressed as base pairs unwound, are shown over a 30-s interval (right). The positions of these records were histogrammed in a single density plot (middle, yellow); strong peaks in this plot indicate locations where the complex pauses, which were not correlated with the location of the AUGs.
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