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The role of a unique chromodomain insertion in a canonical ATP binding domain in eukaryotic Elongation Factor 3 (eEF3) in Saccharomyces cerevisiae

by
Arjun N. Sasikumar
B.E., Birla Institute of Technology - 2007

Thesis Advisor: Terri Goss Kinzy, Ph.D.
Graduate Program in Microbiology & Molecular Genetics

RWJMS Research Tower, Room R-747
Piscataway

Monday, April 29, 2013
1:00 p.m.


Abstract

Translation, the process by which proteins are synthesized based on the information encoded in mRNA, involves initiation, elongation and peptide chain termination reactions. It is mediated by ribosomes and multiple soluble factors, many of which are conserved across bacteria and eukaryotes. During elongation, eukaryotic Elongation Factor 1A (eEF1A; EF-Tu in bacteria) delivers aminoacylated-tRNA to the A-site of the ribosome while eEF2 (EF-G in bacteria) is responsible for the translocation of ribosome along the mRNA. Fungal translation elongation is striking in its absolute requirement for a third factor, the ATPase eEF3. eEF3 binds close to the E-site of the ribosome and has been proposed to facilitate the removal of deacylated tRNA from the E-site. eEF3 has two ATP Binding Cassette (ABC) domains, the second of which carries a unique chromodomain-like insertion hypothesized to play a significant role in its binding to the ribosome. This model was tested in the current study using a mutational analysis of the Sac7 region of the chromodomain. Specific chromodomain mutations result in reduced growth rate as well as slower translation elongation. in vitro analysis suggests that while the chromodomain mutations do not affect the ability of eEF3 to interact with the ribosome, the ATPase activity is severely compromised indicating an allosteric effect on ATP binding/hydrolysis. We also undertook a bioinformatic analysis to study the distribution and conservation of the supposed fungal-specificity of eEF3 across all eukaryotes and found out that, contrary to the current consensus, the presence of eEF3 is not limited to the fungal kingdom. In agreement with its role as a heterodimeric twin cassette ATPase, all the putative eEF3 homologs analyzed had highly conserved ABC domains.


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