Abstract
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.
Highlights
Translation initiationfactoreIF-4Bpromotesthe allow the ribosome to bind and scan toward the initiatAoUr G bindingofmRNAto40 S preinitiation complexes and codon downstream from the 5’-cap [4, 5]. eIF-4F binds to the together with eIF-4A possesRseNsA helicase activity.To m7G cap through the action of its smallest subunit, eIF-4Fa
After dilution with 50 p1 of Expression of eIF-4B Mutant cDNA in COS-1 Cells-The various binding buffer, the beads were centrifugedfor 30 s in a microfuge, and mutant eIF-4B cDNAs were excised from the pSP72 vector by digestion the supernatant was removed and saved
The middle of the protein is rich in Asp, Arg, Tyr, and Gly and is called the DRYG region, ribonucleoside complex (New England Biolabs) and0.3%Nonidet P-40 whereas theC-terminal third is somewhat rich in Serresidues and used for RNA analyses
Summary
Eral Medical Sciences. 8 Supported as a trainee in the National Institutes of Health Training. Digestion of pSP724B with BglII or XbaI RIPA buffer[15].Clarified cell lysates 6-10) werecentrifuged in a followed by religation of the plasmid results ina 39-amino acid (AC39) Beckman TLlOO centrifuge for 20 min at 400,000 x g to pellet riboor 164-aminoacid (AC164)C-terminal truncationof the eIF-4B protein, somes. EIF-4B Bindingto Biotinylated RNA-RNA binding of eIF-4B forms terminal deletion mutants were sequenced directly in the pvSePct7o2r in the 5-100 or HSW subcellular fractions was determined by using by using a primer complementary to the T7 promoter region of the biotinylated mRNA bound to streptavidin-conjugated magnetic beads vector. After dilution with 50 p1 of Expression of eIF-4B Mutant cDNA in COS-1 Cells-The various binding buffer, the beads were centrifugedfor 30 s in a microfuge, and mutant eIF-4B cDNAs were excised from the pSP72 vector by digestion the supernatant was removed and saved. Beginning a t 40-48 h posttransfection, the cells were incubated for 20 min with 1.5 ml of
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