Cloning and sequencing of complementary DNAs encoding the alpha-subunit of translational initiation factor eIF-2. Characterization of the protein and its messenger RNA.
A clone encoding the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha) was isolated from a lambda gt11 expression library of rat brain cDNAs. The fusion protein expressed by the recombinant phage reacts with eIF-2 alpha antiserum except when the serum is preadsorbed with pure eIF-2. The translation of hybrid-selected HeLa cell mRNA produces two proteins which are indistinguishable from authentic HeLa eIF-2 alpha and its phosphorylated form when analyzed by electrophoresis in two-dimensional isoelectrofocusing/sodium dodecyl sulfate-polyacrylamide gels and by partial protease digestion. HeLa cell eIF-2 alpha mRNA migrates as a single band of about 1600 nucleotides. The rat cDNA insert was sequenced, and the region coding for eIF-2 alpha was identified. A human cDNA clone was obtained by hybridization screening with the rat cDNA, and its sequence was determined also. Both rat and human eIF-2 alpha proteins comprise 315 amino acids (36.1 kDa) and differ by only three amino acids. The eIF-2 alpha mRNA is found exclusively in polysomes containing 10 or more ribosomes in exponentially growing HeLa cells. In serum-depleted cells which synthesize eIF-2 and bulk protein more slowly than exponential cells, the level of eIF-2 alpha mRNA is not changed, the average polysome size is reduced to 7, and little or no eIF-2 alpha mRNA is detected in the ribonucleoprotein fraction. These results are consistent with the view that eIF-2 alpha mRNA translation is very efficient compared to other mRNAs in the cell.
D000595:Amino Acid Sequence; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001923:Brain Chemistry; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015852:Eukaryotic Initiation Factor-2; D006801:Humans; D009693:Nucleic Acid Hybridization; D010448:Peptide Initiation Factors / Q000235:genetics*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats
D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D011506:Proteins; D012333:RNA, Messenger; D004247:DNA