A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3.
We have investigated how point mutations in the two ATP-binding motifs (G(463)PNGCGK(469)ST and G(701)PNGAGK(707)ST) of elongation factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae. The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine(463 and 701) and lysine(469 and 707) were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3's binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine(463 and 701) was replaced with valine. The mutant EF-3s did not support cell growth in TEF3-disrupted S. cerevisiae, when each lysine(469 and 707) and glycine(463) was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae.
Yang
H
H
Department of Mycology, Nippon Roche Research Center, Japan.
Hamada
K
K
Terashima
H
H
Izuta
M
M
Yamaguchi-Sihta
E
E
Kondoh
O
O
Satoh
H
H
Miyazaki
M
M
Arisawa
M
M
Miyamoto
C
C
Kitada
K
K
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry; D001665:Binding Sites; D004926:Escherichia coli; D005656:Fungal Proteins; D005982:Glutathione Transferase / Q000096:biosynthesis; D005998:Glycine; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D010445:Peptide Elongation Factors; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C111584:YEF3 protein, S cerevisiae; C012001:polyphenylalanine; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; D000604:Amino Acyl-tRNA Synthetases; D008239:Lysine; D005998:Glycine