Characterization of Bean Eukaryotic Translation Initiation Factor 5: Cloning, Sequencing, and Structure-Function Studies.
Date of Award
Doctor of Philosophy (PhD)
Sue G. Bartlett
Translation initiation is emerging as one of the important mechanisms involved in regulation of gene expression and our understanding of this process in eukaryotes has greatly increased over the past twenty years. Research in translation initiation in plants has lagged behind efforts in the mammalian system and a number of the initiation factors have not been characterized. Eukaryotic initiation factor 5 (eIF5), the protein mediating hydrolysis of the eIF2-bound GTP and the subsequent formation of the 80S ribosome, is among the least characterized initiation factor in plants. This dissertation describes the cloning of the cDNA encoding eIF5 from a bean seed cDNA expression library as well as the sequencing of the cDNA, and the purification of the recombinant protein. Bean eIF5 shares 37-39% identity and 59-60% similarity with rat and yeast eIF5, respectively, with the majority of the conserved residues at the amino-terminal region of each of these proteins. The conserved elements of the carboxy-terminal region of eIF5 include two highly conserved tryptophans and an acidic tail. While eIF5 is considered to function as a GTPase, the G1 and G2 domains at the amino-terminus are the only strictly conserved GTPase domains found in all of the eIF5s and the G1 domain is atypical of the ras-type GTPase superfamily. It is shown that eIF5 is a C$\sb2$C$\sb2$-type zinc-finger protein, binding zinc in a 1:1 molar ratio. Substitution of two of the zinc-binding cysteines with alanine reveals that the zinc ion is an important structural element of eIF5 but it remains unclear what role the zinc-finger may play in eIFS's ribosomal function. However, eIF5 does interact with nucleic acids and this interaction requires the native form of eIF5. In particular, eIF5 will interact with rRNA in an interaction that requires the presence of the carboxy-terminal region in order to occur with full avidity.
Floyd, Zelpha Elizabeth, "Characterization of Bean Eukaryotic Translation Initiation Factor 5: Cloning, Sequencing, and Structure-Function Studies." (1997). LSU Historical Dissertations and Theses. 6483.