Kinetics and mechanism of the association of the bacteriophage T4 gene 32 (helix destabilizing) protein with single-stranded nucleic acids: Evidence for protein translocation

Abstract

We have investigated the association kinetics of the co-operatively binding T4-coded gene 32 (helix destabilizing) protein with a variety of single-stranded homopolynucleotides (both RNA and DNA). Stopped-flow mixing experiments were performed by monitoring the partial quenching of the intrinsic tryptophan fluorescence of the protein upon binding to the nucleic acid under conditions where the nucleic acid concentration is in great excess over the protein concentration. Investigations of the association rate (and rate constants) as a function of solution variables has demonstrated quite different behavior at the extremes of “low” and “high” salt concentration. Under low salt (high binding constant) conditions the non-co-operative association is rate-limiting and we measure a bimolecular rate constant of 3 × 10 6 to 4 × 10 6 m −1 (nucleotide)s −1 (0·1 m-NaCl, 25·0 °C). However, at higher salt concentrations (lower binding constant) a pre-equilibrium involving non-co-operatively bound protein is established, followed by the rate-limiting formation of co-operatively bound protein clusters. Based on these observations we have proposed a mechanism for the formation of co-operatively bound T4 gene 32 protein clusters, under conditions of low binding density, which consists of three steps: (1) pre-equilibrium formation of non-co-operatively bound protein (nucleation); followed by (2) association of free protein to the singly contiguous sites established in the nucleation step, hence forming the first co-operative interactions (growth step); and (3) a redistribution of the growing protein clusters to form the final equilibrium distribution. From comparisons of our experimental values of the forward rate constant for the second step (growth of clusters) with theoretical estimates based on the work of Berg & Blomberg (1976,1978) we infer that the T4 gene 32 protein is able to translocate along singlestranded polynucleotides. The implications of these results for the in vivo action of the T4 gene 32 protein are discussed.