Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes. 1. Irreversible dissociation induced by sodium chloride concentration jumps.
The dissociation kinetics of cooperatively bound bacteriophage T4 gene 32 protein from a variety of single-stranded homopolynucleotides has been investigated by stopped-flow techniques. Irreversible dissociation of the complexes was induced by rapidly increasing the salt concentration and monitoring the increase in tryptophan fluorescence upon dissociation of the gene 32 protein. The dependence of the apparent dissociation rate constant on initial fractional saturation of the nucleic acid lattice as well as the observation of zero-order kinetics when the lattice is initially fully saturated with protein indicates that dissociation occurs only from the ends of protein clusters and not from doubly contiguous molecules. The data for the entire time course are quantitatively fit by a kinetics model specifying irreversible dissociation of only singly contiguously bound protein [Lohman, T.M. (1983) Biopolymers 22, 1697-1713]. This model is used to extract molecular rate constants for the dissociation of isolated, singly contiguously and doubly contiguously bound protein. It is also shown that the polynucleotide specificity observed for the cooperative binding constant, K omega, and the cooperativity itself are intrinsic properties of the dissociation rate of the various complexes.
D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009994:Osmolar Concentration; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D011131:Polyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013604:T-Phages / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D011089:Polydeoxyribonucleotides; D011131:Polyribonucleotides; D014764:Viral Proteins; D012965:Sodium Chloride