Structure-function relationship in Escherichia coli initiation factors. Identification of a lysine residue in the ribosomal binding site of initiation factor by site-specific chemical modification with pyridoxal phosphate.
Incubation of Escherichia coli initiation factor 3 (IF3) with pyridoxal phosphate (PLP) followed by reduction with sodium borohydride resulted in the selective modification and inactivation of this protein. The ribosomal-binding site (RNA-binding site) of IF3 is the target of PLP modification, since (a) the phosphate residue of PLP is required for inactivation; (b) RNA as well as synthetic polynucleotides (especially guanine-containing one) protect IF3 from inactivation; and (c) 30 S, but not 50 S ribosomal subunits, protect IF3 from PLP modification and from inactivation. The incorporation of PLP into IF3 occurred exclusively at lysine residues by reduction of the Schiff bases yielding epsilon-(5'-phosphopyridoxyl)lysine. The PLP-modified lysines were identified by amino acid analysis and sequencing of the PLP-modified peptides. Out of the 20 lysines of the factor, only Lys 2, Lys 5, Lys 99, Lys 112, Lys 166, and an unidentified Lys of the central cluster of the molecule (Lys 86, 87, 90, 91, 96) were found to be modified to varying degrees. The incorporation of 3 to 4 mol of PLP/mol of IF3 is accompanied by a substantial (greater than or equal to 80%) inactivation of this protein; the loss of activity follows apparent first order kinetics, and the inactivation results from the modification of just 1 Lys residue. This essential Lys residue was identified by various criteria to be Lys 112. The identification of an "active region" in the IF3 molecule is emerging from this as well as from other chemical modification studies.
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010448:Peptide Initiation Factors / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D012270:Ribosomes / Q000378:metabolism; D013329:Structure-Activity Relationship
D000596:Amino Acids; D001426:Bacterial Proteins; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D011732:Pyridoxal Phosphate