Circular dichroism and 500-MHz proton magnetic resonance studies of the interaction of Escherichia coli translational initiation factor 3 protein with the 16S ribosomal RNA 3' cloacin fragment.
The RNA helix destabilizing properties of Escherichia coli initiation factor 3 protein (IF3), and its affinity for an evolutionarily conserved sequence at the 3' end of 16S rRNA, led us to examine the details of the protein-nucleic acid interactions upon IF3 binding to the 49-nucleotide 3'-terminal cloacin DF13 fragment of 16S rRNA by studying the circular dichroism (CD) and proton magnetic resonance spectra of the RNA, the protein, and their complex. In a physiological tris(hydroxymethyl)aminomethane buffer, where the interaction is primarily nonionic and sequence specific, addition of IF3 decreases the RNA 268-nm CD peak hyperbolically by 19% to an end point of about one IF3 per RNA strand. The titration curve is best fit by an association constant of (1.80 +/- 0.05) X 10(7) M-1, within the range estimated by a nuclease mapping study of the same system [Wickstrom, E. (1983) Nucleic Acids Res. 11, 2035-2052]. In a low-salt phosphate buffer without Mg2+, where the interaction is primarily ionic and nonspecific, titration with IF3 decreases the peak CD sigmoidally by 35% to an end point of two IF3 per strand. The titration curve is best fit by an intrinsic association constant of (1.7 +/- 0.7) X 10(6) M-1 for each IF3 and a cooperativity constant of 33 +/- 6. In a physiological phosphate buffer lacking Mg2+, the dispersion of aromatic proton magnetic resonance peaks and upfield-shifted methyl proton resonances indicates a high degree of secondary and tertiary structure in the protein. In an equimolar mixture of IF3 and RNA cloacin fragment, several changes in identifiable IF3 and RNA resonances are observed.(ABSTRACT TRUNCATED AT 250 WORDS)
D001430:Bacteriocins / Q000235:genetics*; D001483:Base Sequence; D002942:Circular Dichroism / Q000379:methods; D002989:Cloacin / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3; D011487:Protein Conformation; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D001430:Bacteriocins; D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D002989:Cloacin