Protein Synthesis Initiation Factor eIF-1A Is a Moderately Abundant RNA-binding Protein

Abstract

Eukaryotic initiation factor (eIF) 1A (formerly called eIF-4C) is a small 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 residues and acidic residues at its N and C termini, respectively. Northwestern blotting with a fragment of mRNA indicates that eIF-1A binds RNA. Overexpression of the human eIF-1A cDNA in Escherichia coli and subsequent purification enabled us to prepare large quantities of active factor. The level of eIF-1A in HeLa cells determined by Western immunoblotting 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 may not be limiting for protein synthesis, a conclusion reinforced by the failure of overproduced eIF-1A to stimulate translation rates in transiently transfected COS-1 cells. S1 nuclease protection and primer extension analyses show that eIF-1A mRNA possesses an unusually long 5'-untranslated leader that is very G/C-rich (72%). Unexpectedly, the mRNA is efficiently translated in HeLa cells as judged by polysome profile analyses.

Initiation of Protein Synthesis in Eukaryotic Cells

“…236) as a bridge between other protein factors or between an initiation factor and the ribosome [27]. In subsequent work Northwestern blotting analysis demonstrated strong interaction between recombinant eIFlA and RNA [28] but the specificity of this has yet to be examined.…”

“…eIF3 has long been known to stabilize 43s preinitiation complexes in vitro, and also to be essential for the binding of these complexes to mRNA. The molecular basis of its role in mRNA binding is still not clear, but potentially important interactions have been observed between eIF3 and other initiation factors involved in this step [3, 341. In addition the yeast eIF3 complex binds directly to RNA [29,301, probably via the 62-kDa subunit [30]; surprisingly, this is not a homologue of the 66-kDa polypeptide in mammalian eIF3, which has recently been shown to bind to an mRNA transcript in a Northwestern blot assay [28] (Asano, K., Naranda, T. and Hershey, J. W. B., personal communication).…”

“…236) as a bridge between other protein factors or between an initiation factor and the ribosome [27]. In subsequent work Northwestern blotting analysis demonstrated strong interaction between recombinant eIFlA and RNA [28] but the specificity of this has yet to be examined.eIF3 is a multimeric complex of total molecular mass 500-750 kDa, consisting of at least eight polypeptide chains in mammalian cells and ten polypeptide chains in wheat germ (reviewed [I, 3, 41 W. B., personal communication) has led to the identification of the yeast polypeptides p90, p62, p39 and p16 as PRTI, GCD10, p36 and SUIl, respectively; the yeast p39 subunit is homologous to the p36 subunit of human eIF3. pl6/SUI1 has been identified as an initiation factor previously listed as eIFl [32].…”

Characterization of Yeast Translation Initiation Factor 1A and Cloning of Its Essential Gene

“…The failure to stimulate translation rates by overexpression of the cDNA in transiently transfected mammalian cells suggests that eIF1A is not limiting for protein synthesis (22). However, because of the intrinsic complexity of the mammalian system and the limited ability to manipulate specific gene expression, we decided to study eIF1A function in yeast.…”

“…One of these, eIF1A (formerly eIF-4C), has been purified from both mammalian (3)(4)(5) and plant cells (6) and is essential for maximal in vitro protein synthesis. eIF1A is a small protein (17)(18)(19)(20)(21)(22) kDa) that appears to undergo no post-translational modification reactions (7). The initiation factor is implicated in 80 S ribosome dissociation, stabilizes initiator Met-tRNA i binding to 40 S ribosomal subunits, and facilitates mRNA binding to the 40 S preinitiation complex (2,8).…”