Site-directed mutagenesis of yeast eEF1A. Viable mutants with altered nucleotide specificity.
Site-directed mutants of eEF1A (formerly eEF-1alpha) 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 (residues 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 polyphenylalanine similar to wild type, although with a Km 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.
Cavallius
J
J
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.
Merrick
W C
WC
D001483:Base Sequence; D017931:DNA Primers; D015977:Eukaryotic Initiation Factor-1 / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D017931:DNA Primers; D015977:Eukaryotic Initiation Factor-1; D006160:Guanosine Triphosphate