Molecular characterization of a prokaryotic translation factor homologous to the eukaryotic initiation factor eIF4A.
Initiation of translation involves a complex series of reactions that result in the formation of an initiation complex at the proper start site of the mRNA. These reactions, particularly those that involve the binding of the mRNA to the small subunit of the ribosome, are not fully understood. Here we show that one of the factors (W2) required to reconstitute translation in E. coli is encoded by the deaD gene which harbors 87% amino acid sequence similarly to the eukaryotic (eIF4A). Antibodies against the eukaryotic eIF4A cross-react with the E. coli protein. We describe the overexpression of the W2 protein from recombinant clones and its purification in one step by the use of a His tag at the N-terminus of its sequence. We report a rapid assay for the W2 protein that scores for initiation and elongation programmed by a native mRNA template. The W2 protein promotes initiation programmed by the mRNA that harbors secondary structures. The W2 protein is not required in standard initiation assays programmed by synthetic mRNAs of defined sequence that lack this feature. We conclude that W2 is an important factor for initiation in eukaryotic and prokaryotic cells.
Lu
J
J
Banting and Best Department of Medical Research, University of Toronto, Ont., Canada.
Aoki
H
H
Ganoza
M C
MC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D005057:Eukaryotic Cells / Q000502:physiology; D039601:Eukaryotic Initiation Factor-4A; D005786:Gene Expression Regulation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000235:genetics*; D011119:Polynucleotides / Q000378:metabolism; D011387:Prokaryotic Cells / Q000502:physiology*; D014176:Protein Biosynthesis; D020365:RNA Helicases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D010448:Peptide Initiation Factors; D011119:Polynucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D039601:Eukaryotic Initiation Factor-4A; D020365:RNA Helicases