Determination of the amino acid sequence of rabbit, human, and wheat germ protein synthesis factor eIF-4C by cloning and chemical sequencing.
The small eukaryotic initiation factor (eIF)-4C is implicated in the initiation pathway, where it enhances ribosome dissociation into subunits and stabilizes the binding of the initiator Met-tRNA(i) to 40 S ribosomal subunits. In order to elucidate the function of eIF-4C, its structure has been further characterized. The amino acid sequence of many peptides from rabbit reticulocyte and wheat germ eIF-4C have been determined chemically. From the chemical sequencing of the rabbit protein, it was noted that at least two different eIF-4C molecules were present which differed by conservative substitutions at three positions (2 aspartic acid for glutamic acid switches and 1 valine for isoleucine switch). By the use of unique sequences with low codon degeneracy, primers were used to obtain a polymerase chain reaction product of appropriate size and sequence. This product was then used to isolate full-length coding sequence cDNA clones for human eIF-4C. A similar strategy was used to design PCR primers and then isolate a wheat cDNA clone which lacked the coding region for the first 23 amino acids, but contained a complete 3'-untranslated region. The protein amino acid sequence of wheat germ eIF-4C is 68% identical with the mammalian protein, and, allowing for the most conservative substitutions, the proteins are 76% similar. Both the mammalian and wheat germ proteins are 143 amino acids in length and have molecular weights of about 16,400. A unique feature of eIF-4C is its apparent "polarity" as 9 of the first 15 amino acids are basic while 13 of the last 20 amino acids are acidic. This dipole nature may enable the protein to interact with both the ribosome (perhaps via the rRNA) and other translation initiation factors.
Dever
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Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935.
Wei
C L
CL
Benkowski
L A
LA
Browning
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K
Merrick
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WC
Hershey
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D015977:Eukaryotic Initiation Factor-1; D006801:Humans; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000096:biosynthesis* / Q000737:chemistry*; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D011817:Rabbits; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000378:metabolism
D017931:DNA Primers; D015977:Eukaryotic Initiation Factor-1; D010448:Peptide Initiation Factors; C026022:eukaryotic peptide initiation factor-1A