title: Mutational analysis of the functional domains of the large subunit of the isozyme form of wheat initiation factor eIF4F.
abstract: The isozyme form of plant eukaryotic initiation factor 4F (eIF(iso)4F) contains two subunits: p28, a cap-binding protein, and p86. To identify the functional domains of p86, truncations of the p86 cDNA were made, and the protein was expressed in Escherichia coli and purified. The deletion mutants were tested for the ability to bind the p28 subunit by two methods. In addition, these deletion mutants were evaluated in vitro by the ability to catalyze eIF4A and RNA-dependent ATP hydrolysis and to support polypeptide synthesis. The loss of the ability to bind p28 occurs within the first 90 amino acids of the N terminus and abrogates the ability of p86 to participate in translation initiation and bind to eIF4A, but does not affect ATP hydrolysis. Up to 299 amino acid residues from the C terminus of p86 must be deleted before an effect is observed on the ATP hydrolysis activity. Thus, the p28 binding and ATP hydrolysis activities appear to lie on two separate domains and are functionally uncoupled. In addition, at least a portion of the eIF4A binding domain appears to be in close proximity to the p28 binding domain and is also uncoupled from the ATP hydrolysis activity.
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA. kbrowning@mail.utexas.edu
authors.forename: A M
authors.initials: AM
authors.lastname: Metz
authors.forename: K S
authors.initials: KS
authors.lastname: Browning
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017384:Sequence Deletion; D014908:Triticum
chemical_list: D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors; D000255:Adenosine Triphosphate