Faculty Bibliography

Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with 32P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or 'unwind' mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. Our results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A

Studies were conducted to determine whether encephalomyocarditis virus infection causes proteolytic cleavage of any of the polypeptides which comprise eucaryotic initiation factor 4F. Since no such alterations in the components of the initiation factor were detected, these observations confirmed that the mechanisms whereby encephalomyocarditis virus and poliovirus shut off host translation are different

Previously, we have described an ATP-dependent recognition and binding of mRNA by eukaryotic initiation factors (eIF)-4A, eIF-4B, and eIF-4F (Grifo, J. A., Tahara, S. M., Leis, J. P., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1982) J. Biol. Chem. 257, 5246-5252; Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1983) J. Biol. Chem. 258, 5804-5810). This finding was consistent with other studies which implicated eIF-4A and eIF-4B in binding mRNA to the 40 S ribosomal subunit, an ATP-requiring process. As part of ongoing studies of this step, and, in particular its ATP requirement, we have examined ATPase activity of various initiation factors. In this communication we describe an RNA-dependent ATP hydrolysis catalyzed by eIF-4A and eIF-4F. Although eIF-4B has little or no ATPase activity it can stimulate the RNA-dependent ATPase activity of either eIF-4A or eIF-4F. Similar to the ATP-dependent mRNA binding assay, the RNA-dependent ATPase activity is inhibited by the cap analogue m7GDP when globin mRNA is used as the activator. In addition, a variety of polynucleotides stimulate the ATPase activity of these factors including rRNA, tRNA, poly(U), and poly(A) but not poly(dA). Finally, an attempt has been made to discern whether phosphorylation or ATP hydrolysis is responsible for the ATP-stimulated binding of mRNA by eIF-4A and eIF-4B. We present evidence which is consistent with the interpretation that ATP hydrolysis and not protein phosphorylation correlates with ATP-stimulated binding of mRNA

The ability of homogeneous phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) from pig spleen to phosphorylate eukaryotic initiation factor 2 (eIF-2) was examined. PL-Ca-PK phosphorylated the beta-subunit of eIF-2, whereas myosin light chain kinase (MLCK) and cyclic AMP- and cyclic GMP-dependent protein kinases (cA-PK and cG-PK) did not. PL-Ca-PK could incorporate a maximum of 1.6 mol phosphate/mol eIF-2. The app. Km and Vmax for PL-Ca-PK phosphorylation of eIF-2 were 0.13 microM and 0.02 mumol.min-1.mg enzyme-1, respectively. Phosphoamino acid analysis revealed that incorporation of phosphate into eIF-2 occurred almost exclusively at serine residues. These findings indicate that eIF-2 was an effective substrate for PL-Ca-PK, suggesting that this enzyme may play a role in the regulation of protein synthesis

The translation of poliovirion RNA (polio RNA) in an in vitro fractionated system was much less efficient than that of encephalomyocarditis virion RNA (EMC RNA). In contrast, when polio and EMC RNAs were added to postmitochondrial cell lysates (S10), they were translated with equal efficiency. However, this equality was observed only when high concentrations of S10 were employed; at lower concentrations, polio RNA translation was reduced relative to that of EMC RNA. These results suggest that both the fractionated and S10 systems are limiting in a component that is required for the optimal translation of polio RNA. The elongation rates for EMC and polio RNA translation in the fractionated system were found to be similar, indicating that this component acts at an initiation step. Various components, including excess ribosomal salt wash and postribosomal supernatant of cell lysate, were added to the fractionated system in an effort to identify the slow step more precisely. Of these, only excess ribosomal salt wash specifically stimulated polio RNA translation, suggesting that one or more initiation factors is necessary in unusually large amounts for this mRNA. Various purified initiation factors were tested for the ability to enhance polio RNA translation. Of these, only purified eukaryotic initiation factor 4A had a specific effect. This suggests that polio RNA, in contrast to other mRNAs tested (EMC, reoviral, and globin), may have an unusually low affinity for this initiation factor. The significance of these results is discussed in terms of the methods picornaviruses have evolved for reprogramming the translational machinery of the host cell

A reconstituted reticulocyte translation system originally designed to be deficient in eukaryotic initiation factor 4B (eIF-4B) was used to identify a new activity required for maximal synthesis of rabbit globin. This new activity purifies as a stable, high molecular weight complex by a variety of chromatographic procedures and is termed eIF-4F. The purified globin stimulatory activity also restores translation of capped mRNAs in extracts of poliovirus-infected HeLa cells. Like restoring activity that was obtained as a protein complex by different procedures (Tahara, S. M., Morgan, M. A. and Shatkin, A. J. (1981) J. Biol. Chem. 256, 791-794), eIF-4F includes the 24,000-dalton cap binding protein and major polypeptides of Mr approximately 200,000 and approximately 46,000. The latter component comigrates with eIF-4A by two-dimensional gel electrophoresis and, like eIF-4A, chemically cross-links to the 5'-end of capped mRNA by an ATP-dependent, m7GDP-sensitive reaction. Unlike eIF-4F, cap binding protein of Mr approximately 24,000 isolated by affinity chromatography on m7GDP-Sepharose does not stimulate globin synthesis in the reconstituted system

Host and reovirus mRNAs compete with one another for translation in infected cells. Kinetic analysis has suggested that the site of competition is a message discriminatory initiation factor which must bind to the mRNA before it can interact with the 40S ribosomal subunit. The present communication describes an in vitro assay which can detect message discriminatory activities. A competitive situation is established by using reovirus and globin mRNAs, and then the specificity with which this competition is relieved by added components is measured. Among the various initiation factors surveyed with this assay, two have the properties expected of the mRNA discriminatory factor. These are eukaryotic initiation factor 4A and a 'cap binding protein' complex. Inasmuch as the cap binding protein complex contains a subunit similar or identical to the initiation factor eIF-4A, it seems likely that only one form of the latter factor may be active in vivo. In vitro, both factors relieve competition among both capped and uncapped reovirus mRNAs according to similar hierarchies. These results suggest that some feature other than the m7G cap, such as nucleotide sequence or secondary structure, is recognized by the discriminatory factor

Eukaryotic initiation factor 4A (eIF-4A) has been purified (to apparent homogeneity) from rabbit reticulocyte lysate. It is a single polypeptide accounting for at least 90% of the Coomassie blue staining material when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight was determined by gel electrophoresis under denaturing conditions at two different bisacrylamide to acrylamide ratios, by gel filtration under native conditions and by sedimentation equilibrium at three different protein concentrations. Additional physical properties of the polypeptide were also determined. In an attempt to characterize the function of eIF-4A, a protein specifically required for mRNA translation, an assay was developed which measures the protein-dependent retention of radiolabeled hemoglobin mRNA on nitrocellulose filters. These studies led to the discovery of an ATP-stimulated binding of mRNA which is dependent on the presence of eIF-4A and eIF-4B that also contains the 24,000-dalton cap binding protein. The reaction apparently requires ATP hydrolysis since a nonhydrolyzable analogue of ATP, adenosine 5'-(beta, gamma-imino)triphosphate, does not stimulate mRNA binding and GTP cannot substitute for ATP. In addition, ATP-stimulated binding of mRNA can be inhibited by an analog of the mRNA 5' terminus, m7GMP, suggesting recognition of the capped 5' end of hemoglobin mRNA. Consistent with this suggestion, ATP also stimulated the covalent cross-linking of eIF-4A and eIF-4B to the cap of oxidized reovirus mRNA, an interaction that was inhibited by m7GDP