Site-directed Mutagenesis of Yeast eEF1A

Abstract

Site-directed mutants of eEF1A (formerly eEF-1) were generated using a modification of a highly versatile yeast shuttle vector (Cavallius, J., Popkie, A. P., and Merrick, W. C. (1997) Biochim. Biophys. Acta 1350, 345-358). The nucleotide specificity sequence NKMD (resi-dues number 153-156) was targeted for mutagenesis, and the following mutants were obtained: N153D (DKMD), N153T (TKMD), D156N (NKMN), D156W (NKMW), and the double mutant N153T,D156E (TKNE). All of the yeast strains containing the mutant eEF1As as the sole source of eEF1A were viable except for the N153D mutant. Most of the purified mutant eEF1As had specific activities in the poly(U)-directed synthesis of polyphenyl-alanine similar to wild type, although with a K m for GTP increased by 1-2 orders of magnitude. The mutants showed a reduced rate of GTP hydrolysis, and most displayed misincorporation rates greater than wild type. The mutant NKMW eEF1A showed unusual properties. The yeast strain was temperature sensitive for growth, although the purified protein was not. Second, this form of eEF1A was 10-fold more accurate in protein synthesis, and its rate of GTP hydrolysis was about 20% of wild type. In total, the wild-type protein contains the most optimal nucleotide specificity sequence, NKMD, and even subtle changes in this sequence have drastic consequences on eEF1A function in vitro or yeast viability.