Date of Award

Fall 2013

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Clyde Denis

Abstract

There are still many protein interactions that occur during translation termination that are poorly understood. One of the important termination pathways still under investigation is nonsense-mediated decay (NMD), which rapidly degrades mRNAs that contain a premature stop codon (PTC). I identified that the interaction between Upfl, which is required for NMD, and PAB1 occurs via the RRM1 domain of PAB1 in the yeast Saccharomyces cerevisiae. Determining the role of this interaction during NMD was performed with pulse-chase assays using a PGKlpG mRNA. These assays revealed that the interaction between Upfl and PAB1 is required for a shift from distributive to processive deadenylation, but is not required for decapping or general decay during NMD. These results also revealed that this interaction plays a role in the normal shift in deadenylation mode for non-PTC containing mRNAs from the relatively slow mode of distributive deadenylation to that of rapid processive deadenylation.

I also investigated the components of general termination complexes associated with the translation termination factor eRF1. This analysis was performed using the novel technique of analytical ultracentrifugation with a fluorescence detection system (AU-FDS) on Flag-eRF1 affinity purified extracts. AU-FDS revealed that Flag-eRF1 associates with six distinct complexes that have S values of 20S, 28S, 39S, 57S, 77S, as well as complexes greater than 100S. All of these complexes contained the closed-loop components eIF4E, eIF4G1, and PABl. However, stoichiometric analysis revealed that the complexes greater than 28S were comprised mostly of free ribosomal subunits associated with eRF1. Glucose deprivation and cycloheximide stress treatments revealed that these complexes are likely post-termination complexes rather than pre-initiation or translationally active complexes.

Additional observations from AU-FDS revealed a shifting behavior for the 20S complex during sedimentation, suggesting a shift in shape or composition during the experiment. Also, the abundance and sedimentation profile of eRF3 suggests that it forms a polymer-like structure consistent with a known eRF3 prion characteristic.

These analyses further reveal the interactions involved in the termination process. The application of AU-FDS continues to prove a useful tool for identifying novel protein complexes.

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