# The Structural Dynamics of Translation Initiation

> **NIH NIH R01** · COLUMBIA UNIV NEW YORK MORNINGSIDE · 2022 · $336,173

## Abstract

PROJECT SUMMARY
 The process through which the two-subunit ribosome assembles at the start codon of an mRNA to
initiate protein synthesis is one of the most fundamental and highly regulated steps of gene expression. As
such, initiation serves as the target of numerous small-molecule antibiotics and cellular pathogens. Moreover,
deregulation of initiation is causally linked to viral infections and tumorigenesis in humans. Given all of this,
studies of the molecular mechanism of initiation hold great promise for the identification and characterization of
mechanistic steps that can serve as targets for the development of next-generation antibiotics and other small-
molecule, anti-viral, and anti-cancer drugs that act by modulating translation initiation.
 Despite their promise for molecular medicine, mechanistic studies of initiation remain incredibly
challenging. This is primarily because initiation is an extraordinarily dynamic, multi-step process that proceeds
through a large number of short-lived intermediate states that are very difficult to observe and characterize
using conventional approaches. During initiation, a set of essential initiation factors (IFs) transiently interact
with both ribosomal subunits and a specialized initiator tRNA in order to guide their assembly at the start codon
of the mRNA to be translated. Although evidence suggests that the IFs, ribosomal subunits, and tRNA undergo
functionally important structural rearrangements during this process, very few of these rearrangements have
been directly observed and/or characterized, and for those that have, it has been at very low resolution.
 The long-term goals of this project are to use powerful combinations of reagents and techniques that
are uniquely available in our and our collaborators’ laboratories to overcome the challenges associated with
mechanistic studies of initiation. Specifically, we will use state-of-the-art single-molecule fluorescence
microscopy and cryogenic electron microscopy (cryo-EM), including a pioneering, time-resolved cryo-EM
approach developed by our collaborator, Dr. Joachim Frank, to directly observe and characterize the dynamics
of initiation. These studies will be enabled by a new approach that we have developed for introducing
fluorophores into ribosomes at positions that are highly desirable, but that have thus far remained out of reach.
 In Aim 1, we will investigate the mechanism through which bacterial IF2 transiently binds to a ribosomal
initiation complex (IC) based on the small, 30S, ribosomal subunit (30S IC); determines whether the 30S IC is
carrying an accurately selected initiator tRNA that is properly base-paired to a correctly selected start codon;
and, if so, recruits and facilitates joining of the large, 50S, ribosomal subunit to the 30S IC. In Aim 2, we will
investigate the mechanism through which bacterial IF3 and IF1 ensure the accuracy with which the start codon
is selected during initiation. In Aim 3, we will extend our studie...

## Key facts

- **NIH application ID:** 10457282
- **Project number:** 5R01GM084288-13
- **Recipient organization:** COLUMBIA UNIV NEW YORK MORNINGSIDE
- **Principal Investigator:** Ruben L Gonzalez
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $336,173
- **Award type:** 5
- **Project period:** 2008-12-01 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10457282

## Citation

> US National Institutes of Health, RePORTER application 10457282, The Structural Dynamics of Translation Initiation (5R01GM084288-13). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10457282. Licensed CC0.

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