Human translation initiation relies on the combined activities of numerous ribosome-associated eukaryotic initiation factors (eIFs). The largest factor, eIF3, is an ∼800 kDa multiprotein complex that orchestrates a network of interactions with the small 40S ribosomal subunit, other eIFs, and mRNA, while participating in nearly every step of initiation. How these interactions take place during the time course of translation initiation remains unclear. Here, we describe a method for the expression and affinity purification of a fluorescently-tagged eIF3 from human cells. The tagged eIF3 dodecamer is structurally intact, functions in cell-based assays, and interacts with the HCV IRES mRNA and the 40S-IRES complex in vitro. By tracking the binding of single eIF3 molecules to the HCV IRES RNA with a zero-mode waveguides-based instrument, we show that eIF3 samples both wild-type IRES and an IRES that lacks the eIF3-binding region, and that the high-affinity eIF3-IRES interaction is largely determined by slow dissociation kinetics. The application of single-molecule methods to more complex systems involving eIF3 may unveil dynamics underlying mRNA selection and ribosome loading during human translation initiation.
This research output has an Altmetric Attention Score of 5. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 05 May 2023.
All research outputs
#7,807,540
of 27,609,497 outputs
Outputs from Nucleic Acids Research
#12,611
of 28,685 outputs
Outputs of similar age
#110,038
of 348,667 outputs
Outputs of similar age from Nucleic Acids Research
#217
of 401 outputs
Altmetric has tracked 27,609,497 research outputs across all sources so far. This one has received more attention than most of these and is in the 71st percentile.
So far Altmetric has tracked 28,685 research outputs from this source. They typically receive a little more attention than average, with a mean Attention Score of 7.4. This one has gotten more attention than average, scoring higher than 55% of its peers.
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