Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein.
Protein synthesis initiation factor 4D (eIF-4D) from mammalian cells contains the post-translationally modified lysine derivative hypusine. A highly purified preparation of the protein from rabbit reticulocytes was subjected to chemical and enzymatic cleavage, and a large number of overlapping peptides were resolved by high performance liquid chromatography and sequenced. Two mixed 14-base DNA probes were synthesized based on suitable amino acid sequences and were used to screen a human cDNA library in lambda gt11. A cDNA insert containing eIF-4D encoding sequences was identified and a 558-base pair EcoRI-PstI fragment was sequenced. Northern blot hybridization of HeLa cell RNA shows a single size class (1.2 kilobase) of mRNA. The DNA encodes a protein comprising 154 residues with a mass of 16,703 daltons. Human eIF-4D matches all of the rabbit peptides sequenced, extending from residue 9 to 154 except for Cys-129 which is Ser in the rabbit protein. The residue modified to hypusine is identified as Lys-50 and the amino terminus is blocked. eIF-4D possesses rather little secondary structure in the amino-terminal two-thirds of the protein, but the carboxyl-terminal third is rich in alpha helices.
Smit-McBride
Z
Z
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Dever
T E
TE
Hershey
J W
JW
Merrick
W C
WC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000032:analysis / Q000235:genetics*; D011487:Protein Conformation; D016601:RNA-Binding Proteins; D011817:Rabbits; D000097574:Eukaryotic Translation Initiation Factor 5A
D004247:DNA; D008239:Lysine; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine