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Fluorescently-tagged human eIF3 for single-molecule spectroscopy.

Alex G Johnson^1,2, Alexey N Petrov³, Gabriele Fuchs⁴

¹Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA.

Nucleic Acids Research

|November 15, 2017

Summary

Researchers developed a fluorescently-tagged human translation factor, eIF3, to study its interactions with mRNA. Single-molecule tracking revealed how eIF3 binds to specific RNA structures, offering insights into translation initiation.

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Area of Science:

Molecular Biology
Biochemistry
Cell Biology

Background:

Human translation initiation is a complex process involving multiple eukaryotic initiation factors (eIFs).
The largest initiation factor, eIF3, is an 800 kDa complex crucial for ribosome association and mRNA interactions during initiation.
The dynamic interactions of eIF3 during translation initiation are not fully understood.

Purpose of the Study:

To develop a method for expressing and purifying a fluorescently-tagged human eIF3 complex.
To investigate the binding kinetics and dynamics of eIF3 with mRNA using single-molecule techniques.
To elucidate the role of eIF3 in mRNA selection and ribosome loading during translation initiation.

Main Methods:

Expression and affinity purification of a fluorescently-tagged human eIF3 complex.
In vitro interaction studies of tagged eIF3 with Hepatitis C Virus (HCV) IRES mRNA and 40S ribosomal subunit complexes.
Single-molecule binding analysis using zero-mode waveguides to track eIF3-RNA interactions.

Main Results:

The fluorescently-tagged eIF3 dodecamer was structurally intact and functional in cell-based assays.
Tagged eIF3 demonstrated interaction with HCV IRES mRNA and the 40S-IRES complex in vitro.
Single-molecule tracking revealed that eIF3 samples different IRES RNA structures, with high-affinity binding characterized by slow dissociation kinetics.

Conclusions:

A functional, fluorescently-tagged human eIF3 complex was successfully generated.
Single-molecule biophysics provides a powerful approach to study the dynamics of eIF3-RNA interactions.
Understanding eIF3 dynamics offers insights into mRNA selection and ribosome loading in human translation initiation.