Reticulocytes initiation factor from

To determine which of the initiation factors was responsible for the resumption of globin ENA synthesis, the purified initiation factors from reticulocytes were added one by one (4 ) These studies suggested that when initiation factor eIP-4B(lF-M3)was present in limiting quantities, viral ENA outcompetes cellular mENA for translation. When the factor is present in excess, both messages are translated. 

To determine the reason for the lack of YSY mRNA translation, purified initiation factors from reticulocytes were added one by one to the inactive extracts and the ability of the extract to support YSV mRNA translation was examined. Only one initiation factor, eIE-4B, could restore YSY mRNA translation, as shown in Figure 5. This is the same factor that Golini at (47) found could restore the translation of cellular mRNA when in competition with viral mRNA. 

Cell extracts were prepared from cells 2 hr (a, b and e) and 3 hr (c, d and f) after infection as described in Figure 2. Fifty m I incubations contained 50 m-M amino acids-methionine, 120 mM KOAc, 2.5 12M Mg (OAc), other components previously described, and no additions (a and o), 1 M.1 of rabbit reticulocyte initiation factors (b and d 30 oP protein), prepared as described (22), or 1 mM 7"Hiethylguanosine-5 -inonophosphate (e and f). 

Benne, R., and Hershey, J. W. B., 1978, The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes, J. Biol. Chem. 253 3078. 

The use of the specific initiation factor from rabbit reticulocytes chromato-graphically purified on DAE-cellulose and phosphocellulose demonstrated the stimulation of hemoglobin mRNA translation but was not effective with the... 

Globin is synthesized in reticulocytes (see Chap. 1, Prob. 1.1). which have no nucleus and therefore cannot utilize transcriptional and other potential modes of control. Control of globin synthesis from the pool of globin-enriched mRNA is geared to the concentration of hemin (Fe(III)-protoporphyrin]. which has the ability to inactivate a translational inhibitor of protein synthesis. The inhibitor is a protein kinase that phosphorylates and inactivates one of the initiation factors involved in initiation of translation. When the concentration of hemin is high, it binds to a regulatory subunit of the kinase and. as a result, initiation of globin synthesis can proceed. 

Why does an anemia result from iron deficiency When an individual is deficient in iron, the reticulocytes do not have sufficient iron to produce heme, the required prosthetic group of hemoglobin. When heme levels are low, the eukaryotic initiation factor eIE2 (see Fig. 16.22) is phosphorylated, and inactive. Thus, globin mRNA cannot be translated because of the lack of heme. This results in red blood cells with inadequate levels of hemoglobin for oxygen delivery, and an anemia. 

Interestingly, in reticulocyte lysates, the effect of ds RNA is similar to that resulting from heme deficiency (63), that is, polypeptide chain initiation is inhibited. (For an extensive discussion, see chapter 10 of this book).. The ds RNA appears to induce a protein kinase which phosphorylates initiation factor eIF-2 (63). Experiments in our laboratory (M. Jaye, unpublished data) have failed to detect the presence of any phosphorylated components in the crude initiation factor preparation after virus infection corresponding to those elicited in the presence of ds RNA. 

Brown, B., and Ehrenfeld, E., 1980, Initiation factor preparations from poliovirus infected cells restrict translation in reticulocyte lysates. Virology 103 327. 

Ernst, V., Levin, D. H., Leroux, A., and London, I. M., 1980, Site-specific phosphorylation of the a-subunit of eukaryotic initiation factor eIF-2 by the heme-regulated and double-stranded RNA-activated eIF-2a kinases from rabbit reticulocyte lysates, Proc. Natl. Acad. Sci. USA 77 1286. 

Initiation of protein synthesis in mammalian cells proceeds by a complex process whereby the assembly of mRNA, the ribosome, and initiator met-tRNAf into an initiation complex is catalyzed by a group of proteins called initiation factors. By definition, these proteins are not required for polypeptide chain elongation. A detailed discussion of this process, and the role of individual initiation factors can be found in this volume (Kaempfer, 1984), or in other recent reviews (Benne and Hershey, 1978 Jagus et al., 1981) which also contain pertinent references. Nine initiation factors have been highly purified from rabbit reticulocytes, and still other factors have been described which may serve auxiliary functions, or indeed may qualify as initiation factors in their own right. A very brief (and oversimplifed) description of the role of the nine, characterized initiation factors is listed below, along with the step in initiation complex formation in which each participates ... 

The resolution of this apparent discrepancy was provided by an independent discovery by Sonenberg et al. (1978) that a small polypeptide (Mr 24,000) which specifically bound the m G cap structure at the 5 end of capped mRNAs, was present in preparations of both eIF-3 and eIF-4B purified from rabbit reticulocytes. The name capbinding protein (CBP) was given to this polypeptide. The activity which restored VSV mRNA translation by poliovirus-infected cell extracts, and which had previously been ascribed to eIF-4B was subsequently shown to be due to CBP that had copurified with eIF-4B (Trachsel et al., 1980). Subsequent comparisons of eIF-4B from uninfected and poliovirus-infected HeLa cells showed no differences in the number of molecules per cell, molecular size, or extent of covalent modification of this initiation factor (Duncan et al., 1983). Similarly, the eIF-3 preparations isolated from poliovirus-infected cells which were inactive in vitro, likely contained both eIF-3 and CBP, since the... 

However, when mammalian Met tRNA is formylated with the appropriate bacterial system, the loaded transfer RNA can serve as initiator. A protein factor necessary for initiation, found in reticulocytes and referred to as M3, seems to have properties similar to those of the IF3 factor in bacteria. M2 A and M2B protein factors have also been isolated from reticulocytes and are said to be indispensable for the AUG-dependent binding of Met tRNA. A third binding factor M has also been discovered in reticulocytes its exact role is not clear. 

One approach was to prepare extracts of poliovirus-infected HeLa cells and to assay for an inhibitor of initiation by addition to a reticulocyte lysate translation reaction. The reticulocyte lysate had been well-characterized as a system capable of efficient initiation in vitro. A potent inhibitor of initiation was indeed found in the cytoplasm of poliovirus-infected cells, which was not present in similar preparations from uninfected HeLa cells (Hunt and Ehrenfeld, 1971), and the inhibitor was subsequently identified as double-stranded poliovirus RNA (Ehrenfeld and Hunt, 1971). In order to qualify as a specific inhibitor of host cell protein synthesis, however, it was necessary to demonstrate that viral protein synthesis was immune to the inhibitory effects of the putative shut-off factor. When double-stranded RNA was put to this test, no specificity between cellular and viral protein synthesis could be demonstrated (Celma and Ehrenfeld, 1974) rather, all protein synthesis was inhibited equally. In addition, concentrations of viral double-stranded RNA required to... 

---