# Dynamics of eukaryotic translation initiation and its control

> **NIH NIH R01** · STANFORD UNIVERSITY · 2021 · $471,947

## Abstract

PROJECT SUMMARY (30 lines)
Initiation of translation in eukaryotic organisms is rate limiting and highly regulated. The process
involves a score of protein factors guiding 40S subunits to the 5’ untranslated region (5’UTR) of
an mRNA, and subsequent directional scanning to the first start codon, followed by 60S subunit
joining and transition to elongation. The mechanistic basis for this process remains unclear.
During the prior funding period we developed single-molecule approaches to provide a real-time
dynamic perspective to yeast translation initiation. Here we build on our methodological
developments over the past funding period to provide combined structural and dynamic view of
how initiation occurs in yeast and humans. In Aim 1 we focus on how 40S subunits are prepared
and then join the 5’ end of an mRNA and leverage our labeled factors to understand the
dynamics by which the eIF4F complex guides this process in collaboration with mRNA and
helicases; we then will explore the directional scanning to the start codon and delineate the role
of 5’ UTR length sequence and structure and how 3’ terminal proteins and RNA modulate the
process. In Aim 2, we investigate how start codon recognition occurs through 60S subunit
joining and the transition to elongation. We determine the conformation/composition signals in
start codon recognition, and why eIF5B induced transition to elongation is slow, and whether
initiation factors linger during elongation. We will correlate slow events with known in vivo
measurements and probe the consequences of queuing in scanning. In Aim 3, we investigate
alternative initiation pathways, such as leaky scanning, near cognate start sites and upstream
open reading frame translation, termination and re-initiation. For all aims, we merge dynamic
and biochemical investigations with structural characterization of intermediates by cryoEM
guided by our single-molecule observations. The proposed research is buttressed by strong
collaborations on the various systems to support biophysical and structural analysis, reagent
preparation, and in vivo correlation. The results of this proposal will provide deep and broad
view of the interplay of mRNA sequence and structure and the pathways of translation initiation,
providing a foundation to understand translational control and its mis-regulation in disease.

## Key facts

- **NIH application ID:** 10145015
- **Project number:** 5R01GM113078-06
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** JOSEPH D PUGLISI
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $471,947
- **Award type:** 5
- **Project period:** 2016-09-01 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10145015

## Citation

> US National Institutes of Health, RePORTER application 10145015, Dynamics of eukaryotic translation initiation and its control (5R01GM113078-06). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10145015. Licensed CC0.

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