Mutations in a GTP-binding motif of eukaryotic elongation factor 1A reduce both translational fidelity and the requirement for nucleotide exchange.
A series of mutations in the highly conserved N(153)KMD(156)GTP-binding motif of the Saccharomyces cerevisiae translation elongation factor 1A (eEF1A) affect the GTP-dependent functions of the protein and increase misincorporation of amino acids in vitro. Two critical regulatory processes of translation elongation, guanine nucleotide exchange and translational fidelity, were analyzed in strains with the N153T, D156N, and N153T/D156E mutations. These strains are omnipotent suppressors of nonsense mutations, indicating reduced A site fidelity, which correlates with changes either in total translation rates in vivo or in GTPase activity in vitro. All three mutant proteins also show an increase in the K(m) for GTP. An in vivo system lacking the guanine nucleotide exchange factor eukaryotic elongation factor 1Balpha (eEF1Balpha) and supported for growth by excess eEF1A was used to show the two mutations with the highest K(m) for GTP restore most but not all growth defects found in these eEF1Balpha deficient-strains to near wild type. An increase in K(m) alone, however, is not sufficient for suppression and may indicate eEF1Balpha performs additional functions. Additionally, eEF1A mutations that suppress the requirement for guanine nucleotide exchange may not effectively perform all the functions of eEF1A in vivo.
Carr-Schmid
A
A
Department of Molecular Genetics, University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School, Ohio 44106, USA.
Durko
N
N
Cavallius
J
J
Merrick
W C
WC
Kinzy
T G
TG
D001665:Binding Sites; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D009154:Mutation; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D020648:Peptide Elongation Factor 1; D006160:Guanosine Triphosphate