Alleviating ribosomal stalls: functional characterization of conserved Drg GTPases

To meet cellular demands for precise location-dependent protein concentrations, translation is regulated in both temporally and spatially. As one way to implement such control, translating ribosomes temporarily halt or pause on the mRNA upon which they are translating to allow for control of the timing, localization, and abundance of the associated nascent proteins. This thesis reports the conserved function of a new class of translational GTPases, Developmentally Regulated GTP-binding (Drg) proteins, that mediate this translational regulation. Analyses reveal that bacterial Obg GTPases are distant orthologs of eukaryotic and archaeal Drg proteins (Rbg proteins in yeast) and that Drg factors share conserved functions to alleviate stalling in ribosomes that were induced to stall in vivo. Bacterial Obg rescues growth and translation defects in both S. cerevisiae and human cells lacking their endogenous Drg factors. This function is dependent on GTP hydrolysis, as GTPase-deficient mutants fail to rescue stalling. Further in vitro biochemical characterization reveals that Rbg1 unexpectedly hydrolyzes ATP. Both human Drg1 and E. coli ObgE stimulate peptidyl transfer, the core catalytic function of ribosomes, giving mechanistic insight into the conserved function of Drg GTPases. Proteomic and transcriptomic analyses reveal that eukaryotic Drg factors target ribosomes synthesizing a wide repertoire of proteins, many of which are related to nuclear, secretory, and mitochondrial functions. Given their broad role in proteostasis, Drg proteins are implicated in cancer progression, and a high-throughput screen was performed to identify small molecules that inhibit Drg-mediated translation function using a dual-fluorescent stalling reporter mRNA. These findings position conserved Drg proteins as translation factors with a conserved role in modulating ribosome dynamics and maintaining proteostasis across all three domains of life.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Hawk II, Christopher William

Contributors dc:contributor

• Jin, Hong
• Nair, Satish
• Kalsotra, Auinash
• Wu, Nicholas

• Ribosome
• Translation
• Drg

Statement dc:rights

• Copyright 2025 Christopher Hawk II

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/130136

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F130136