Protein-DNA Interactions Studied at Sedimentation Equilibrium

Abstract

The sedimentation equilibrium (SE) technique has been in use for nearly 80 years. 1 As a result, the literature describing applications of the technique is vast. Excellent reviews have been published during the past decade 2-6 and earlier work is also a valuable resource. 7-10 The analysis of macromolecule-ligand interactions developed over the same period and is the subject of a similarly large literature. Here, we will present results representative of recent work in our laboratories that demonstrate the promise and the challenges inherent in SE analysis of protein-nucleic acid interactions and will briefly discuss advances that are likely to prove valuable in future studies. We have used the methods described here in studies of sequencespecific and non-specific DNA interactions of transcription factors, DNA repair proteins and components of the DNA-replication machinery. In addition, these approaches are likely to be useful for the analysis of interactions that modulate chromatin structure and the protein-RNA interactions of ribosome assembly, tRNA charging and mRNA maturation. We hope that this review will be useful to workers in all these fields. At SE a system of macromolecules will have a radial distribution of concentrations described by Equation (1).Here, the summation is over all species, i; c(r) is the observed concentration at radial position r, c i,0 the concentration of the ith species at the reference position r 0 (typically close to the meniscus), the reduced molecular weight, σ i is equal to M i (1Ϫν ෆi ρ)ω 2 /2RT, where M i is the molecular weight, ν ෆi the partial specific volume