Xin Wang

Date of Award

Spring 2013

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

Clyde L Denis

Abstract

The identification of the components involved in translational complexes has relied primarily on in vitro studies. Determining which proteins associate together in these complexes, under what conditions they do so, and how the composition of the complexes change under different conditions have became the key issues of in vivo studies. After a one-step affinity purification, using a novel technique of analytical ultracentrifugation with a fluorescence detection system (AU-FDS) I have identified a 77S monosomal translational complex in the yeast Saccharomyces cerevisiae. Major components of the 77S complex include the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G1/eIF4G2 and PAB1. The abundance of 77S complexes decreased with translational defects and following the stresses that cause translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions, I observed that the stress of glucose depletion affected translation initiation primarily by operating through a pathway involving eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KC1) and robust heat shock acted at other steps.

Subsequently, I have determined the absolute abundance changes of all factors identified in the 77S complex during different steps of the translation process. To address this question thoroughly, I also analyzed six new proteins shown to be present in the 77S complex: SBP1, SLF1, PUB1, SUP35, SUP45 and SSD1. By comparing the ratios of AU-FDS/A230, I found only 56% of monosomal complexes contained eIF4E and only 34% contained eIF4G during translation elongation, implying that not all 77S complexes were in the closed-loop structure. During termination, eRF1 and SBP1 to a lesser extent were found to be enriched, suggesting a role for SBP1 in the termination process, possibly in translational repression. SLF1, SSD1, and SBP1 abundances also changed at initiation, suggesting specific roles for these proteins in this process.

This stoichiometric analysis gives us a dynamic view of how the composition of the translational complex changes in vivo. This application of stoichiometric analysis using AU-FDS should prove widely adaptable to the identification of protein complexes in other biological systems.

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