Post-translational modifications of eIF5A and its bacterial ortholog EF-P: Structural and functional analogy

Tuesday, October 09, 2012 — Poster Session I
1:00 p.m. – 3:00 p.m	Natcher Conference Center, Building 45	NIDCR	BIOCHEM-11

Authors

• JH Park
• XH Liang
• MH Park

Abstract

Eukaryotic initiation factor 5A (eIF5A) and bacterial elongation factor P (EF-P) represent one of few universally conserved pairs of translation factors. Both enhance ribosomal methionyl-puromycin synthesis in vitro and have also been implicated in translation elongation. The two proteins share similarity in their amino acid sequences and crystal structures. EF-P was proposed to undergo beta-lysylation by two enzymes, YjeA and YjeK. We used the polycistronic vector, pST39, to overexpress E. coli EF-P alone, or together with YjeA and YjeK in E. coli. Unmodified EF-P or alpha-lysyl EF-P or beta-lysyl-EF-P was produced using pST39/EF-P or pST39/EF-P/YjeA, or pST39/EF-P/YjeA/YjeK. Native EF-P was found to contain ~ 1 mol of beta-lysine per mol of protein. Both native EF-P and recombinant EF-P were active in stimulating methionyl-puromycin synthesis, whereas unmodified EF-P, alpha-lysyl-EF-P and EF-P mutant protein (K34A) were inactive, supporting the importance of beta-lysylation in EF-P activity. In a parallel study, we constructed pST39 vectors encoding human or yeast eIF5A alone, eIF5A plus deoxyhypusine synthase (DHPS), eIF5A plus DHPS plus deoxyhypusine hydroxylase (DOHH) and overexpressed unmodified eIF5A, deoxyhypusinated eIF5A and hypusinated eIF5A, respectively. Highly purifed recombinant eIF5A containing hypusine displayed comparable activity to native eIF5A purified from mammalian tissue, in the methionyl-puromycin synthesis assay.

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