Faculty Bibliography

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