# Disentangling conformational and compositional heterogeneity

> **NIH NIH F32** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2020 · $64,926

## Abstract

ABSTRACT
It has recently been discovered that “specialized” eukaryotic ribosomes enriched or depleted in certain core
ribosomal proteins (RPs) preferentially translate groups of mRNA sequences, and moreover that these groups
of mRNAs often relate to specific cellular pathways ​(Shi et al., 2017)​. This link between ribosome
compositional heterogeneity and genome regulation has been studied by mass spectrometric and sequencing
analyses of ribosomes and mRNA sequences isolated from distinct ribosome populations. However, simple
rules linking “specialized” ribosomes and sequence preferences have not yet emerged. Work by our
collaborator Maria Barna’s lab showed that the presence or absence of RPS25/eS25, a substoichiometric RP
located near the mRNA exit tunnel on the 40S subunit of the eukaryotic ribosome, affects translation of ~150
genes. Our hypothesis is that this core RP imparts recognition of mRNAs by altering the structure and
conformational dynamics of the ribosome. To address this question, we will isolate and characterize ribosomes
with or without RPS25/eS25 by cryoEM.
Due to its fundamentally single particle nature, cryoEM is an optimal method for examining large
heterogeneous macromolecular machines. However, at present the end result of cryoEM analysis is a single
structure reflecting the average of all contributing conformations. We propose to develop a new class of model
that incorporates the concept of a dynamic structure with conformational heterogeneity. We will collaborate
with the developers of cisTEM to parameterize flexibility of a structural model and to iteratively refine model
motions alongside the canonical structure during refinement of cryoEM maps. We will refine flexible models of
ribosomes with and without RPS25/eS25 using this new functionality to discover a link between structure and
function, revealing the atomic mechanisms of translation specificity and implicating a general mechanism for
genome regulation by ribosome composition.
In summary, we will develop new structure refinement capabilities for cryoEM that encode structural flexibility
and apply them to the discovery of differences in flexibility between ribosomes preferential to different mRNA
sequences. This study will be a proof of principle for the discovery of biologically relevant structural motions by
single particle cryoEM, and the expanded ribosome and mRNA models will suggest a mechanism for genome
regulation at the stage of protein synthesis.

## Key facts

- **NIH application ID:** 10049965
- **Project number:** 5F32GM133129-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Iris Diane Young
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $64,926
- **Award type:** 5
- **Project period:** 2019-09-17 → 2022-09-16

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10049965

## Citation

> US National Institutes of Health, RePORTER application 10049965, Disentangling conformational and compositional heterogeneity (5F32GM133129-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10049965. Licensed CC0.

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