In vitro analysis of eIF5A binding to the ribosome using fluorescence anisotropy: insights from alanine scanning mutants of eIF5A

Abstract

The translation factor 5A (eIF5A) is highly conserved in Archaea and eukaryotes and is essential for cell viability. This is the only protein known to contain the amino acid residue hypusine, essential for eIF5A function, generated by a post-translational modification. Although eIF5A has been extensively involved in several cellular processes, it was only recently determined a role for eIF5A in protein synthesis, more specifically, in the elongation cycles. It was suggested that, during translation elongation, eIF5A enhances the peptide bond formation of specific amino acid sequences. To improve the description and understanding of the mechanism of eIF5A in translation, it is necessary to determine the points of interaction in both ribosome and eIF5A. Our laboratory, in collaboration with Dr. Christopher S. Fraser and Dr. John W. Hershey's laboratory, has previously developed a fluorescence anisotropy assay to measure the kinetics of eIF5A binding to the ribosome. In this project, we propose to use this fluorescence anisotropy assay to investigate the kinetics of eIF5A-ribosome complex formation of a series of new yeast eIF5A mutants recently generated in our laboratory. These new eIF5A mutants were generated based on clustered charged-to-alanine (alanine scanning) residues on eIF5A surface, so we can identify the residues important for eIF5A physicial interaction with the ribosome. We generated 13 clustered charged-to-alanine mutants of eIF5A and tested for their ability to replace wild type eIF5A. Out of these 13 mutants, 7 showed no difference in growth capacity, 5 displayed a temperature-sensitive phenotype and 1 was not able to replace wild type eIF5A in the cell. Therefore, the use of the fluorescence anisotropy assay to measure eIF5A binding to the ribosome will help elucidate whether the residues targeted in these clustered charged-to-alanine mutants of eIF5A are directly involved in eIF5A physical contact to the ribosome. (AU)