Stopped-Flow Fluorescence Kinetic Study of Protein Sliding and Intersegment Transfer in the Target DNA Search Process

Kinetic characterizations of protein translocation on DNA are nontrivial because the simultaneous presence of multiple different mechanisms makes it difficult to extract the information specific to a particular translocation mechanism. In this study, we have developed new approaches for the kinetic investigations of proteins' sliding and intersegment transfer (also known as "direct transfer") in the target DNA search process. Based on the analytical expression of the mean search time for the discrete-state stochastic model, we derived analytical forms of the apparent rate constant k(app) for protein-target association in systems involving competitor DNA and the intersegment transfer mechanism. Our analytical forms of k(app) facilitate the experimental determination of the kinetic rate constants for intersegment transfer and sliding in the target association process. Using stopped-flow fluorescence data for the target association kinetics along with the analytical forms of k(app), we have studied the translocation of the Egr-1 zinc-finger protein in the target DNA association process. Sliding was analyzed using the DNA-length-dependent k(app) data. Using the dependence of k(app) on the concentration of competitor DNA, we determined the second-order rate constant for intersegment transfer. Our results indicate that a major pathway in the target association process for the Egr-1 zinc-finger protein is the one involving intersegment transfer to a nonspecific site and the subsequent sliding to the target. (C) 2013 Elsevier Ltd. All rights reserved.