PROTEIN-SYNTHESIS INITIATION-FACTOR EIF-1A IS A MODERATELY ABUNDANT RNA-BINDING PROTEIN

Eukaryotic initiation factor (eIF) 1A (formerly called eIF-4C) is a sm all protein that promotes dissociation of 80 S ribosomes into subunits , stabilizes methionyl-tRNA binding to 40 S ribosomal subunits, and is required for the binding of mRNA to ribosomes. The sequence of eIF-1A derived from its cloned cDNA possesses a high frequency of basic resi dues and acidic residues at its N and C termini, respectively. Northwe stern blotting with a fragment of mRNA indicates that eIF-1A binds RNA . Overexpression of the human eIF-1A cDNA in Escherichia coli and subs equent purification enabled us to prepare large quantities of active f actor. The level of eIF-1A in HeLa cells determined by Western immunob lotting is 0.01% of total protein, which corresponds to 0.2 molecules of eIF-1A/ribosome. The moderate abundance means that eIF-1A is equal to or in excess of native 40 S subunits and suggests that the factor m ay not be limiting for protein synthesis, a conclusion reinforced by t he failure of overproduced eIF-1A to stimulate translation rates in tr ansiently transfected COS-1 cells. S1 nuclease protection and primer e xtension analyses show that eIF-1A mRNA possesses an unusually long 5' -untranslated leader that is very G/C-rich (72%), Unexpectedly, the mR NA is efficiently translated in HeLa cells as judged by polysome profi le analyses.