Structure-function analysis of Escherichia coli translation initiation factor IF3: tyrosine 107 and lysine 110 are required for ribosome binding.
Translation initiation factor IF3 is required for peptide chain initiation in Escherichia coli. IF3 binds directly to 30S ribosomal subunits ensuring a constant supply of free 30S subunits for initiation complex formation, participates in the kinetic selection of the correct initiator region of mRNA, and destabilizes initiation complexes containing noninitiator tRNAs. The roles that tyrosine 107 and lysine 110 play in IF3 function were examined by site-directed mutagenesis. Tyrosine 107 was changed to either phenylalanine (Y107F) or leucine (Y107L), and lysine 110 was converted to either arginine (K110R) or leucine (K110L). These single amino acid changes resulted in a reduced affinity of IF3 for 30S subunits. Association equilibrium constants (M-1) for 30S subunit binding were as follows: wild-type, 7.8 x 10(7); Y107F, 4.1 x 10(7); Y107L, 1 x 10(7); K110R, 5.1 x 10(6); K110L, < 1 x 10(2). The mutant IF3s were similarly impaired in their abilities to specifically select initiation complexes containing tRNA(fMet). Toeprint analysis indicated that 5-fold more Y107L or K110R protein was required for proper initiator tRNA selection. K110L protein was unable to mediate this selection even at concentrations up to 10-fold higher than wild type. The results indicate that tyrosine 107 and lysine 110 are critical components of the ribosome binding domain of IF3 and, furthermore, that dissociation of complexes containing noninitiator tRNAs requires prior binding of IF3 to the ribosomes.
De Bellis
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Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595.
Liveris
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Goss
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Ringquist
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Schwartz
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D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000737:chemistry*; D005454:Fluorescence Polarization; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism*
D004269:DNA, Bacterial; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D014443:Tyrosine; D012343:RNA, Transfer; D008239:Lysine