Protein synthesis initiation factor eIF-1A is a moderately abundant RNA-binding protein.
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.
Wei
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CL
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
MacMillan
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SE
Hershey
J W
JW
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D015977:Eukaryotic Initiation Factor-1; D006801:Humans; D015151:Immunoblotting; D007668:Kidney; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014162:Transfection
D000906:Antibodies; D017931:DNA Primers; D018076:DNA, Complementary; D015977:Eukaryotic Initiation Factor-1; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C026022:eukaryotic peptide initiation factor-1A