The effect of initiation factor IF-3 on Escherichia coli ribosomal subunit association kinetics

• Published in: The Journal of biological chemistry 1981-07, Vol.256 (13), p.6600-6607
• Main Authors: Chaires, J B, Pande, C, Wishnia, A
• Format: Article
• Language: English
• Subjects: Escherichia coli; Escherichia coli - metabolism; Kinetics; Macromolecular Substances; Mathematics; Peptide Initiation Factors - metabolism; Prokaryotic Initiation Factor-3; Ribosomal Proteins - metabolism; Ribosomes - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)69032-0

IF-3 strikingly inhibits the rate of association of 30 S and 50 S subunits to form 70 S ribosome, at all Mg2+ concentrations between 1 and 18 mM, in 60 mM KCl, pH 7.8, at 26 degrees C. The rate of formation of 70 S is determined from the increase in light scattering intensity in stopped flow experiments in which, typically, largely dissociated 30 S-50S mixtures in 2 mM Mg2+ buffer are jumped against high [Mg2+] buffers containing either no or 1-2 eq of IF-3. The curves can be analyzed using two reactions (the anti-association model): 30 S + 50 S (formula see text) Both forward rates are large: in the physiologically important range, 3-6 mM Mg2+, k1 increases from 0.4 to 15 X 10(6) M-1 s-1, while k2 ranges from 3 to 18 X 10(6) M-1 s-1. The kinetic curves have a characteristic shape: an early spurt of 70 S particle formation while IF-3 and 50 S particles are competing for the large initial pool of free 30 S subunits, and a slower phase in which 70 S formation is controlled by the release of 30 S subunits from the 30 S.IF-3 complexes (k-2 runs between 0.05 and 0.17 s-1). The addition of a third reaction, 30 S.IF-3 + 50 S (formula see text) 70 S + IF-3, is not required for an adequate fit, changes the values of k2 slightly, and of k-2 not too much, and yields values of k-3 (0-20 X 10(3) M-1 s-1) probably too small to play a significant physiological role in initiation of protein synthesis unaided by other interactions. The "anti-association" effect of IF-3 on ribosomal subunits is clear-cut. The "pro-dissociation" effect on 70 S ribosomes remains to be demonstrated.