Eukaryotic protein synthesis initiation factor 4B (eIF‐4B) is an 80,000 dalton polypeptide which is essential for the binding of mRNA to ribosomes. A highly purified preparation of eIF‐4B from HeLa cells was subjected to enzymatic cleavage and amino‐terminal amino acid sequence analysis. Degenerate oligonucleotide probes were used to isolate a 3851 bp cDNA encoding eIF‐4B from a human cDNA library. The DNA encodes a protein comprising 611 residues with a mass of 69,843 daltons. The amino‐terminal domain of eIF‐4B contains a consensus RNA binding domain present in a number of other RNA binding proteins. Expression of eIF‐4B in transfected COS‐1 cells yielded a polypeptide which reacted with anti‐eIF‐4B antiserum and comigrated with purified eIF‐4B. Expression of eIF‐4B in COS‐1 cells resulted in a general inhibition of translation, possibly due to a 50‐fold eIF‐4B overproduction.
Five initiation factors, eIF-2, eIF-3, eIF-4A, eIF-4B, and eIF-5, were purified from human HeLa cells. Methods of protein fractionation and assays for initiation factors which had been developed for rabbit reticulocytes were found to be suitable for HeLa factors. The initiation factors from HeLa cells are similar to or indistinguishable from the corresponding rabbit reticulocyte factors with respect to specific activities, molecular weights as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subunit structure. The molecular weight of eIF-3 particles from both species is about 410000 as determined by equilibrium sedimentation analytical centrifugation. The partial protease fragmentation patterns of corresponding proteins also are similar and indicate that the primary sequences of the factors are related in the two species. Antisera raised in goats against rabbit eIF-3 and human eIF-2, eIF-4A, and eIF-4B cross-react with the cognate factors from both species. On the basis of immunoblotting techniques, eIF-4A is highly conserved, eIF-2 alpha, eIF-3, and eIF-4B are somewhat less conserved, and eIF-2 beta is the least conserved of the proteins examined. The functional, structural, and immunological results are all consistent with the view that initiation factors from different mammalian cells are very similar.
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