Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

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Abstract

Eukaryotic translation initiation factor ₃ (eIF₃) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF₃ functional variants spanning its ₅ essential subunits using an in vitro-reconstituted system. Mutations throughout eIF₃ disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF₃a CTD and eIF₃b/i/g significantly slow mRNA recruitment, and mutations within eIF3_b/i/g destabilize eIF₂•GTP•Met-tRNA_i binding to the PIC. Using model mRNAs lacking contacts with the ₄0S entry or exit channels, we uncover a critical role for eIF₃ requiring the eIF₃a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF₃a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

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Colin Echeverría Aitken

Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

Competing interests
No competing interests declared.
Petra Beznosková

Laboratory of Regulation of Gene Expression, Institute of Microbiology ASCR, Prague, Czech Republic

Competing interests
No competing interests declared.
Vladislava Vlčkova

Laboratory of Regulation of Gene Expression, Institute of Microbiology ASCR, Prague, Czech Republic

Competing interests
No competing interests declared.
Wen-Ling Chiu

PharmaEssentia Corporation, Taipei, Taiwan

Competing interests
No competing interests declared.
Fujun Zhou

Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

Competing interests
No competing interests declared.
Leoš Shivaya Valášek

Laboratory of Regulation of Gene Expression, Institute of Microbiology ASCR, Prague, Czech Republic

For correspondence
valasekl@biomed.cas.cz

Competing interests
No competing interests declared.
Alan G Hinnebusch

Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

For correspondence
ahinnebusch@nih.gov

Competing interests
Alan G Hinnebusch, Reviewing editor, eLife.

"This ORCID iD identifies the author of this article:" 0000-0002-1627-8395
Jon R Lorsch

Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

For correspondence
jon.lorsch@nih.gov

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-4521-4999

Funding

National Institutes of Health (Intramural Research Program)

Colin Echeverría Aitken
Wen-Ling Chiu
Fujun Zhou
Alan G Hinnebusch
Jon R Lorsch

Wellcome (090812/B/09/Z)

Colin Echeverría Aitken
Leoš Shivaya Valášek

Centrum of Excellence of the Czech Science Foundation (P305/12/G034)

Colin Echeverría Aitken
Leoš Shivaya Valášek

Leukemia and Lymphoma Society (5199-12)

Colin Echeverría Aitken

National Institutes of Health (GM62128)

Jon R Lorsch

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.