Characterization of the p33 subunit of eukaryotic translation initiation factor-3 from Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry 1999-03, Vol.274 (13), p.8546-8553
• Main Authors: Hanachi, P. (University of California, Davis, CA.), Hershey, J.W.B, Vornlocher, H.P
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; ARN MENSAJERO; ARN MESSAGER; ARN RIBOSOMAL; ARN RIBOSOMIAL; BINDING PROTEINS; BINDING SITES; Cell Division - genetics; CLONACION; CLONAGE; CLONING; Cloning, Molecular; Eukaryotic Initiation Factor-3; Fungal Proteins - chemistry; Fungal Proteins - genetics; GENE; GENES; MESSENGER RNA; Molecular Sequence Data; NUCLEOTIDE SEQUENCE; Peptide Initiation Factors - chemistry; Peptide Initiation Factors - genetics; Polyribosomes - genetics; PROTEINAS AGLUTINANTES; PROTEINE DE LIAISON; RIBOSOMAL RNA; RIBOSOMAS; RIBOSOME; RIBOSOMES; RNA BINDING DOMAINS; RNA RECOGNITION; RNA RECOGNITION MOTIFS; RNA, Messenger - metabolism; RNA, Ribosomal - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - genetics; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; SECUENCIA NUCLEOTIDICA; Sequence Analysis, DNA; Sequence Deletion - genetics; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; STRUCTURAL GENES; TIF35 GENE; TRANSLATION FACTORS
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.13.8546

Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNA i and mRNA. The molecular mechanism by which eIF3 exerts these functions is incompletely understood. We report here the cloning and characterization of TIF35 , the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3. p33 is an essential protein of 30,501 Da that is required in vivo for initiation of protein synthesis. Glucose repression of TIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni 2+ affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity.