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

Abstract

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-nmCD 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) .times. 107 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 IF3s per strand. The titration curve is best fit by an intrinsic association constant of (1.7 .+-. 0.7) .times. 106 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. In the aromatic region, resonances of Tyr-107, which is part of the ribosomal binding site of IF3, exhibit a shift or broadening, while resonances of the nonessential Tyr-75 are unaffected. The imino proton resonance of G22-C27 at the top of the cloacin fragment hairpin stem shifts upfield underneath the G18-C31 peak, and the imino proton resonances of the adjacent A20-U29 and U19-G30 base pairs in the center of the hairpin broaden extensively, implicating these base pairs in IF3 RNA complex formation. Both effects were seen previously upon heating the RNA in the absence of protein [Heus, H. A., van Kimmenade, J. M. A., van Knippenberg, P. H., Haasnoot, C. A. G., de Bruin, S. H., and Hilbers, C. W. (1983) J. Mol. Biol. 170, 939-956]. The broadening was maintained in the presence of 1 M NaCl, suggesting that this particular interaction may not be ionic. The CD and NMR results may be correlated with nuclease and chemical probe results to yield a model wherein IF3 interacts with the most highly conserved nucleotides of the clofin fragment.