Eukaryotic translation initiation factor 2 (eIF2) has been implicated in the selection of the AUG codon as the start site for eukaryotic translation initiation, since mutations in its three subunits in yeast that allow the recognition of a UUG codon by the anticodon of the initiator Met-tRNAMet have been identified. All such mutations in the beta subunit of eIF2 (eIF2β) mapped to a region containing a putative zinc finger structure of the C2-C2 type, indicating that these sequences could be involved in RNA recognition. Another feature of eIF2β that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three repeats of seven lysine residues which are highly conserved in other species. We show here the ability of eIF2β, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro. Through a deletion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C2-C2 motif. Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run. We further show that only one run of lysine residues is sufficient for the in vivo function of eIF2β, probably through charge interaction, since its replacement by arginines did not impair cell viability, whereas substitution for alanines resulted in inviable cells. mRNA binding, but not GTP-dependent initiator Met-tRNAMet binding, by the eIF2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit.

The β Subunit of Eukaryotic Translation Initiation Factor 2 Binds mRNA through the Lysine Repeats and a Region Comprising the C2-C2 Motif

The study of double-stranded RNA (dsRNA) encompasses a variety of fields. Basic research in this area has contributed to a greater mechanistic understanding of gene induction, tumor cell growth arrest, the establishment of antiviral states, and immunomodulation. Because of the possible clinical value of these molecules, physicians are now exploring the use of synthetic dsRNA to treat patients with cancer, HIV-1 disease, and immune dysfunction. Continued studies of the mechanisms of action of dsRNA are likely to suggest an even wider scope of clinical applications.

Cellular response to double-stranded RNA.

By means of affinity chromatography of lysates from Krebs II ascites cells and rabbit reticulocytes on Sepharose-heparin an active fraction of initiation factors has been obtained.



The fraction is eluted from the column at 350 mM KCl using a linear gradient and displays a number of activities, i.e. binding of Met-tRNAMetf to form a ternary complex with GTP; transferring this complex to 40-S subunits in an A-U-G-independent step and finally coupling of the 40-S initiation complex to the 60-S subunit, a reaction which is completely A-U-G-dependent. Moreover, Met-tRNA is bound into the P-site as is indicated by its puromycin sensitivity. The method is very suitable for large-scale preparation. Further purification and characterization of the factors have been carried out on DEAE-cellulose and phosphocellulose columns.



Evidence is presented that the polysomes present in a lysate that has been passed through the Sepharose-heparin column can only complete their nascent chains, initiation of new polypeptides is completely dependent on addition of initiation factors.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1977.tb11548.x

Initiation of Protein Synthesis in Eukaryotes

The role of the β-subunit of initiation factor eIF-2 in initiation complex formation

Binding of ATP to eukaryotic initiation factor 2. Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAMetf.

Binding of ATP and messenger RNA by the β-subunit of eukaryotic initiation factor 2

In addition to binding messenger RNA molecules at specific sequences, eukaryotic initiation factor 2 (eIF-2) also binds to double-stranded RNA (dsRNA). The dsRNA is a powerful inhibitor of initiation of eukaryotik translation, causing the inactivation of eIF-2, but in the presence of certain mRNA templates, dsRNA fails to establish inhibition. Such mRNA templates bind to eIF-2 with higher affinty than does dsRNA, while globin mRNA, a template sensitive to inhibition, binds with lower affinity.



Here, the nature of the interaction between dsRNA and eIF-2 was studied by examining both the binding of eIF-2 to Penicillium chrysogenum dsRNA molecules carrying 32P label at their 5′ ends, and the ability of eIF-2 to protect such label against pancreatic ribonuclease digestion. The results reveal binding sites for eIF-2 at the 5′ ends, as well as throughout internal regions of the dsRNA molecule. At least 15 molecules of eIF-2 can be accommodated on a 3000-base molecule of P. chrysogenum dsRNA eIF-2 protects a 105-base-pair 5′-terminal fragment in dsRNA against digestion, but exhibits no noticeable preference for the 5′ ends. By contrast, eIF-2 fails to protect label at the 5′ ends of denatured dsRNA molecules, even though it binds to them at internal sites more avidly than to native dsRNA.



Binding of eIF-2 to dsRNA is not restricted to specific sequences: eIF-2 binds with equal affinity to the synthetic dsRNA sequence, poly(rI·rC). The data support the interpretation that eIF-2 recoginzes the A conformation in dsRNA rather than sequence. Apparently binding of eIF-2 at sites spaced 200 base pairs apart prevents relaxation of the intervening length of the double helix, thereby stabilizing the dsRNA molecule against ribonuclease attack.



These results show that, even though dsRNA and mRNA compete in their binding to eIF-2, the structural features recognized by eIF-2 in these RNA species are distinct.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1984.tb08564.x

The nature of the interaction of eukaryotic initiation factor 2 with double‐stranded RNA

Relief of cytotoxicity and enhancement of interferon inducer activity of double-stranded RNA by eukaryotic initiation factor 2.

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Binding of ATP and messenger RNA by the β-subunit of eukaryotic initiation factor 2

The nature of the interaction of eukaryotic initiation factor 2 with double‐stranded RNA

Relief of cytotoxicity and enhancement of interferon inducer activity of double-stranded RNA by eukaryotic initiation factor 2.