# The mechanism of mRNA recruitment to the human ribosome.

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2021 · $331,272

## Abstract

PROJECT SUMMARY
The initiation stage of eukaryotic translation determines: (1) which mRNA is recruited to the ribosome for
translation; and (2) which initiation codon is selected as the translation start site. Changes in physiological
stimuli reprogram the translation machinery to alter preferential recruitment of mRNAs to the ribosome
and which start site is selected. Genome wide analysis has shown that the regulation of these events is
far more extensive than previously appreciated. Significant progress has been made in elucidating the
mechanism of initiation codon selection, but the mechanism of mRNA recruitment to the ribosome and its
regulation remains poorly understood. This knowledge gap persists because of difficulties in determining
which intermediate step(s) in the initiation pathway function as kinetic checkpoints to control mRNA
recruitment to the ribosome. To date, initiation pathway intermediates have been identified on the basis of
their thermodynamic stability which must be enough to withstand traditional assays. These approaches
take minutes to hours to perform, and/or require cross-linking agents to stabilize them, but most
intermediates prior to initiation codon selection occur on the sub-second to second time scale. To
determine how mRNAs are selected for translation, it will be necessary to generate new assays that can
monitor the formation of pathway intermediates in real-time. To overcome this bottleneck, we have
developed ensemble and single-molecule fluorescence-based assays that can observe the rate of mRNA
recruitment to the human ribosome in real time. A highly purified complete reconstituted system that we
have developed will form the cornerstone of this proposal. Models that we generate with this system will
be tested using translation assays in cell-free extracts and intact cells. The long-term objective of this
proposal is to understand the mechanism by which alterations in initiation factor availability and post-
translational modification reprograms the translational apparatus to control which mRNAs are recruited to
the ribosome in response to physiological stimuli.
Aim 1. Characterize the mechanism by which human eIF4F binds to mRNA. Despite over three
decades of research, the molecular details to explain how human eIF4F binds to the cap-proximal region
of an mRNA is still poorly defined. The primary barrier that has prevented progress is the inability to
generate recombinant full-length human eIF4G to carry out sophisticated kinetic assays. Our preliminary
data has unexpectedly revealed that eIF4E possesses both cap-dependent and cap-independent
functions in promoting mRNA binding to eIF4F.
Aim 2. Identify and characterize kinetic checkpoints that control mRNA selection for translation.
Early initiation pathway intermediates that control mRNA recruitment to the 40S subunit are poorly
defined. It is possible that the accommodation of a mRNA into the mRNA entry channel of the 40S subunit
commits it for translatio...

## Key facts

- **NIH application ID:** 10150851
- **Project number:** 5R01GM092927-12
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** Christopher S Fraser
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $331,272
- **Award type:** 5
- **Project period:** 2010-04-05 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10150851

## Citation

> US National Institutes of Health, RePORTER application 10150851, The mechanism of mRNA recruitment to the human ribosome. (5R01GM092927-12). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10150851. Licensed CC0.

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