# Alternative mechanisms of different stages in eukaryotic translation

> **NIH NIH R01** · SUNY DOWNSTATE MEDICAL CENTER · 2021 · $323,000

## Abstract

Initiation is the most complex, tightly regulated stage of eukaryotic protein synthesis. The process begins with
formation of the 48S initiation complex (48S IC) at the initiation codon of mRNA. First, the 43S preinitiation
complex (43S PIC) comprising the 40S ribosomal subunit, the eIF2•GTP•Met-tRNAMeti ternary complex and
eukaryotic initiation factors eIF3, eIF1 and eIF1A binds to the cap-proximal region of mRNA in a step that is
mediated by eIFs 4A, 4B and 4F, which cooperatively unwind the cap-proximal region, allowing for 43S PIC
association. The 43S PIC then scans downstream to the initiation codon where it forms the 48S IC with the
established codon-anticodon interaction. Scanning on structured mRNAs additionally requires the DExH-box
protein DHX29 that binds directly to 40S subunits. eIFs 1 and 1A play key roles in ensuring the fidelity of initiation
codon selection. Initiation codon recognition triggers dissociation of eIF1, eIF5-induced hydrolysis of eIF2-bound
GTP and release of Pi. Subsequent joining of a 60S subunit is promoted by the translational GTPase eIF5B.
Initiation on some viral mRNAs is mediated by an internal ribosome entry site (IRES). IRESs are highly structured
RNA elements that promote 5’-end independent recruitment of the 40S subunit via non-canonical interactions
with the 40S subunits and/or eIFs. Dysregulation of translation initiation is frequently observed in devastating
diseases and is therefore becoming a focus for chemo-therapeutic intervention. Although the factors required for
initiation have been identified, and their principal roles determined, important details concerning its molecular
mechanism, regulation and alternative modes remain unknown. Characterization of these details is therefore a
priority. We have reconstituted the entire translation cycle in vitro, which gives us the unique opportunity to
address critical gaps in understanding of the mechanisms of mammalian initiation and the regulation of
translation using biochemical and complementary biophysical and cell biology approaches. Aim 1 will concern
characterization of the mechanisms by which DHX29 promotes scanning, eIF5B stabilizes Met-tRNAiMet on the
40S subunit and both factors influence initiation codon selection. In Aim 2, we will focus on investigating the
mechanisms of physiologically important initiation with Leu-tRNALeu at CUG codons, and on non-AUG triplets
during repeat-associated non-AUG (RAN) translation, which occurs on expansion repeats in mRNAs transcribed
from genes that are responsible for severe neurodegenerative diseases. Aim 3 is devoted to elucidation of the
molecular mechanism of initiation on the IRES located in the 5'UTR of Cricket paralysis virus RNA, which has a
unique structure and that our preliminary data suggest can use novel mechanisms for initiation. Aim 4 concerns
the cellular function and mechanism of action of Schlafen14, a novel endoribonuclease that binds 80S ribosomes
and cleaves rRNA and ribosome-bound mRNA...

## Key facts

- **NIH application ID:** 10161790
- **Project number:** 5R01GM097014-08
- **Recipient organization:** SUNY DOWNSTATE MEDICAL CENTER
- **Principal Investigator:** CHRISTOPHER Ulrich Tristram HELLEN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $323,000
- **Award type:** 5
- **Project period:** 2012-05-15 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10161790

## Citation

> US National Institutes of Health, RePORTER application 10161790, Alternative mechanisms of different stages in eukaryotic translation (5R01GM097014-08). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10161790. Licensed CC0.

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