Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): demonstration that eIF3 interacts with eIF5 in mammalian cells.
Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit protein complex that plays an essential role in the binding of the initiator methionyl-tRNA and mRNA to the 40S ribosomal subunit to form the 40S initiation complex. cDNAs encoding all the subunits of mammalian eIF3 except the p42 subunit have been cloned in several laboratories. Here we report the cloning and characterization of a human cDNA encoding the p42 subunit of mammalian eIF3. The open reading frame of the cDNA, which encodes a protein of 320 amino acids (calculated Mr35 614) has been expressed in Escherichia coli and the recombinant protein has been purified to homogeneity. The purified protein binds RNA in agreement with the presence of a putative RNA binding motif in the deduced amino acid sequence. The protein shows 33% identity and 53% similarity with the Tif35p subunit (YDR 429C) of yeast eIF3. Transfection experiments demonstrated that polyhistidine-tagged p42 protein, transiently expressed in human U20S cells, was incorporated into endogenous eIF3. Furthermore, eIF3 isolated from transfected cell lysates contains bound eIF5 indicating that a specific physical interaction between eIF5 and eIF3 may play an important role in the function of eIF5 during translation initiation in eukaryotic cells.
Bandyopadhyay
A
A
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University,Jack and Pearl Resnick Campus, Bronx, New York, NY 10461, USA.
Maitra
U
U
D000595:Amino Acid Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D039621:Eukaryotic Initiation Factor-3; D040061:Eukaryotic Initiation Factor-5; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid