Mutations in the NKXD consensus element indicate that GTP binds to the gamma-subunit of translation initiation factor eIF2.

Initiation factor eIF2 binds GTP and promotes the binding of methionyl-tRNA to ribosomes. Biochemical and sequence evidence suggests that the GTP might bind to either the beta- or gamma-subunit of eIF2. Mutations were made in the NKXD consensus elements found in both subunits and individual mutant forms were overexpressed in transiently transfected COS-1 cells. The effect on the translational efficiency of a reporter mRNA for dihydrofolate reductase was monitored. Mutations in the gamma-subunit cause severe repression of protein synthesis, whereas those in the beta-subunit are only mildly inhibitory. The results support the view that GTP binds exclusively to the gamma-subunit.

Two structural domains of initiation factor eIF-4B are involved in binding to RNA.

Translation initiation factor eIF-4B promotes the binding of mRNA to 40 S preinitiation complexes and together with eIF-4A possesses RNA helicase activity. To elucidate structural features involved in its function, a series of internal and C-terminal deletions, as well as point mutations, were constructed in the eIF-4B cDNA. The mutated cDNAs were expressed in transiently transfected COS-1 cells, and mutant forms of the factor were overproduced up to about 25-fold over endogenous eIF-4B levels. Inhibition of dihydrofolate reductase (DHFR) synthesis by high levels of eIF-4B variants was determined in vivo, and the binding of the eIF-4B forms to biotinylated RNA was measured in vitro. The results indicate that the N-terminal region containing the RNA binding motif with its RNP1 and RNP2 consensus elements is sufficient for inhibition of DHFR synthesis. Deletion of the RNP1 sequence abrogates RNA binding, but amino acid substitutions at conserved residues do not always inhibit RNA binding. Deletion of the DRYG domain near the middle of eIF-4B results in inhibition of RNA binding, but not of DHFR synthesis. Up to 164 residues of the C terminus are not required for RNA binding, but removal of 226 or more residues completely inhibits RNA binding, perhaps by the loss of two arginine-rich regions. The results suggest that both the RNA recognition motif and the arginine-rich region are required for stable RNA binding but that both are not necessary for in vivo inhibition of protein synthesis.