Saccharomyces cerevisiae elongation factor 2. Genetic cloning, characterization of expression, and G-domain modeling.
The elongation factor 2 (EF-2) genes of the yeast Saccharomyces cerevisiae have been cloned and characterized with the ultimate goal of gaining a better understanding of the mechanism and control of protein synthesis. Two genes (EFT1 and EFT2) were isolated by screening a bacteriophage lambda yeast genomic DNA library with an oligonucleotide probe complementary to the domain of EF-2 that contains diphthamide, the unique posttranslationally modified histidine that is specifically ADP-ribosylated by diphtheria toxin. Although EFT1 and EFT2 are located on separate chromosomes, the DNA sequences of the two genes differ at only four positions out of 2526 base pairs, and the predicted protein sequences are identical. Genetic deletion of each gene revealed that at least one functional copy of either EFT gene is required for cell viability. Messenger RNA levels of yeast EF-2 parallel cellular growth and peak in mid-log phase cultures. The EF-2 protein sequence is strikingly conserved through evolution. Yeast EF-2 is 66% identical to, and shares over 85% homology with, human EF-2. In addition, yeast and mammalian EF-2 share identical sequences at two critical functional sites: (i) the domain containing the histidine residue that is modified to diphthamide and (ii) the threonine residue that is specifically phosphorylated in vivo in mammalian cells by calmodulin-dependent protein kinase III, also known as EF-2 kinase. Furthermore, yeast EF-2 also contains the Glu-X-X-Arg-X-Ile-Thr-Ile "effector" sequence motif that is conserved among all known elongation factors, and its GTP-binding domain exhibits strong homology to the G-domain of Escherichia coli elongation factor Tu (EF-Tu) and other G-protein family members. Based upon these observations, we have modeled the G-domain of the deduced EF-2 protein sequence to the solved crystallographic structure for EF-Tu.
Perentesis
J P
JP
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Phan
L D
LD
Gleason
W B
WB
LaPorte
D C
DC
Livingston
D M
DM
Bodley
J W
JW
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000235:genetics*; D010750:Phosphoproteins / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D019204:GTP-Binding Proteins