Submission Date

5-4-2026

Document Type

Paper- Restricted to Campus Access

Department

Biology

Adviser

Dale Cameron

Committee Member

Dale Cameron

Committee Member

Cory Straub

Committee Member

Rebecca Evans

Department Chair

Denise Finney

Project Description

Protein function depends on folded structure, with disruptions to normal folding causing an altered function.  Prions are misfolded proteins that gain the function of transmitting their misfolded conformation to their natively-folded counterparts, prompting aggregation.  One factor that may increase the frequency of protein misfolding is cotranslational stalling in the ribosome, where translation may slow or pause with the nascent polypeptide exposed to the cellular environment.  Without the full amino acid sequence and related stabilizing interactions, proteins may be more prone to misfolding.

To determine whether ribosome stalling during translation of aggregation-prone protein domains in yeast influences the rate of protein misfolding, we engineered plasmids to encode the yeast prion-forming domain of Sup35 or a pathogenically-expanded polyglutamine tract with putative stall-inducing sequences downstream of the aggregation-prone domains. Thus, ribosome stalling should leave aggregation-prone nascent polypeptides exposed to the cellular environment.  We employed a yeast prion formation assay to examine misfolding rates of aggregation-prone domains with and without ribosome stalling.

Additionally, we investigated the role of the ribosome-associated chaperone complex (RAC) in early protein folding.  RAC putatively mediates this process via recruiting Hsp70 to the nascent polypeptide.  Using yeast strains with and without functional RAC, we assess how cotranslational chaperone activity modulates the effects of ribosome stalling.  Our results support prior findings that cotranslational stalling and RAC deletions independently promote prion formation.  However, conjoint RAC deletion and cotranslational stalling caused a decrease in prion formation relative to RAC deletion alone, suggesting that these interruptions to protein synthesis are not additive.

Comments

This work was funded by the National Institutes of Health NIGMS under award number R15GM119081 to DMC.

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