Date of Award

Spring 2011

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Clyde Denis

Abstract

The primary components of the translation initiation complex have been identified by a variety of techniques. However, it is likely that all components of the translation initiation complex are still not fully discovered. Identifying new components should lead to a better understanding of the translation process and how it is regulated. Using mass spectrometric studies, we have identified 41 non-ribosomal proteins and non-translation initiation factors as possible components of the translation initiation complex. To determine which of these proteins are in the translation complex, we applied analytical ultracentrifugation with fluorescent detection system (AU-FDS) to detect this complex. Following a one-step affinity purification with Flag-PAB1 using strains carrying translation factors and specific mRNA tagged with GFP, we identified the 78S translation complex that contains all of the major components expected of the translation initiation complex: mRNA, eIF4E, eIF4G1/eIF4G2, PAB1, 40S and 60S ribosomal components. Using GFP fused to about half of the 41 putative novel proteins of the components of the 78S translation complex, we were able to identify at five new proteins, SBP1, SLF1, PUB1, SUP35 and SSD1 as being part of this complex. SBP1 had previously been shown to be a component of stress granules formed following glucose depletion and SLF1 to be associated with translational process. PUB1 could bind to ARE and STE sequence. SUP35 was also reported in the 80S translation complex in 2008. SSD1 had a global effect on translation, and is found associate with the 5'-UTR of CLN2 mRNA. Components of the eIF3 and eIF2alpha complex were also found to be part of the 78S complex, although formaldehyde cross-linking was required to stabilize their association with this complex. These results confirm the utility of AU-FDS for charactering the constitution of multi-subunit complexes and identified 5 new proteins in the 78S translation complex.

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