Protein synthesis in yeast. Structural and functional analysis of the gene encoding elongation factor 3.
The yeast translational elongation factor 3 (EF-3) stimulates EF-1 alpha-dependent binding of aminoacyl-tRNA by the ribosome. The requirement for EF-3 is unique to fungi; a functional analog has not been found in prokaryotes or other eukaryotes. We have isolated and characterized the structural gene, YEF3, that encodes EF-3. The YEF3 gene is present in one copy/haploid genome and is essential for vegetative growth. DNA sequence analysis revealed that the YEF3 gene contains an open reading frame of 1044 codons. The deduced amino acid sequence contains two repeats of a nucleotide-binding motif, which is similar to the nucleotide-binding consensus sequences of hydrophilic, membrane-associated ATPases. EF-3 catalyzes ATP hydrolysis in a ribosome-dependent manner. A modified assay procedure has been developed that allows measurement of the ATP hydrolytic activity of EF-3 in cell-free extracts without interference by other nucleotide hydrolyase activities. Using this modified assay, we have shown that the wild-type YEF3 gene restores heat stable EF-3 activity in a yeast strain containing a temperature-sensitive EF-3. Introduction of the YEF3 gene on a high copy number plasmid into yeast strains increases the ribosome-dependent ATPase activity. The level of EF-3 protein is also increased 3-5-fold. Elevated EF-3 protein levels did not cause a significant increase in EF-1 alpha and EF-2 protein. Yeast strains containing elevated EF-3 protein levels are more sensitive to the aminoglycoside antibiotics hygromycin and paromomycin. These drugs are known to increase translational errors. This observation suggests that EF-3 may indirectly affect translational accuracy.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
M G
MG
Sandbaken
J A
JA
Lupisella
B
B
DiDomenico
K
K
Chakraburtty
D000595:Amino Acid Sequence; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005656:Fungal Proteins; D005800:Genes, Fungal; D005838:Genotype; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004271:DNA, Fungal; D005656:Fungal Proteins; D010445:Peptide Elongation Factors; D029701:Saccharomyces cerevisiae Proteins; C111584:YEF3 protein, S cerevisiae