September 22, 2019  |  

Fluorescently-tagged human eIF3 for single-molecule spectroscopy.

Authors: Johnson, Alex G and Petrov, Alexey N and Fuchs, Gabriele and Majzoub, Karim and Grosely, Rosslyn and Choi, Junhong and Puglisi, Joseph D

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.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Journal: Nucleic acids research
DOI: 10.1093/nar/gkx1050
Year: 2018

Read Publication

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.