XXXV Reunião Anual da SBBqResumoID:9072


Structural analysis of eucariotic translation initiation factor 5A (eIF5A)

Rangel, S.M.1; Olhê, C.A.1; Zanelli, C.F1.; Apponi, L.H.1; Frigieri, M.C.1; Muniz, J.R.C.2; Garratt; R.C.2 and Valentini, S.R.1



Depto. de Ciências Biológicas – Fac. Ciências Farmacêuticas – Universidade Estadual Paulista – UNESP 1; Centro de Biotecnologia Molecular e Estrutural/CEPID-Fapesp, Instituto de Física de São Carlos - USP 2

The putative translation initiation factor 5A (eIF5A) is highly conserved from archebacteria to mammals and essential for cell viability. This factor is the only cellular protein known to contain the amino acid residue hypusine, generated by a spermidine-dependent post-translational modification of a specific lysine residue. This factor has been associated with translation initiation, cell proliferation, nucleocytoplasmatic transport and mRNA decay, but the biological role of eIF5A is still unclear. Three temperature-sensitive alleles of TIF51A (tif51A-1, tif51A-2 and tif51A-3) were characterized and described in our laboratory. In order to understand the biological significance of specific residues of eIF5A, different strategies were used to generate new mutants of this protein. Initially, the structure of yeast eIF5A was modeled based on the solved structure of Leishmania mexicana eIF5A homologue, which was used to predict spacial localization of modified residues in the new mutants. These alleles were obtained by random mutagenesis using EMS or mutagenic PCR and site-direct mutagenesis to mutate specific residues. It was generated 23 different mutants so far, among which 6 demonstrated temperature-sensitive phenotype and 9 were unviable. These mutants have distinct amino acid substitutions in different points of eIF5A and also show different degrees of temperature-sensitivity. Protein levels were evaluated at the non-permissive temperature by western blot and only one conditional mutant produced stable eIF5A at the restrictive temperature, showing that most of the residues modified in these mutants are determinant for structure stability. These new described TIF51A alleles will be very useful for further genetic analysis involving eIF5A which might help understand the function of this essential factor.

Suported by FAPESP and CAPES