Generation and production of single-cysteine mutants of human eIF5A-2 to be used on ribosome binding assays

Abstract

The translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes and is essential for cellular viability. This is the only protein that contains the amino acid hypusine, essential for the function of eIF5A and it is generated by a posttranslational modification. After being involved in several cellular processes, only recently was defined a better function for eIF5A in the process of protein synthesis, specifically during the elongation step of translation. In humans, there are two isoforms of eIF5A. eIF5A-1 is the most widely produced in different tissues. Furthermore, eIF5A-2 has been implicated in the development of the tumor process. To improve understanding and description of the mechanism of eIF5A in translation, it is necessary to determine how is the connection between eIF5A and the ribosome and what kind of ribosomal complex does eIF5A bind. Recent results from our laboratory have shown a direct binding of eIF5A-1 and the large subunit of the ribosome, and the hypusine is required for this interaction. To better understand the functional differences between eIF5A-1 and eIF5A-2, it is proposed the generation of a single cysteine mutant of eIF5A-2 to be used in different assays that reveals the ribosomal complex to which eIF5A binds as well as the sites of interaction. The results of this project will contribute significantly to the understanding of the functional differences of human eIF5A isoforms. (AU)