Kinetic characterization of the sequence-specific binding of prokaryotic and eukaryotic gene regulatory proteins

Abstract

Proteins can bind to specific DNA sequences at rates that are orders of magnitudes above the diffusion-controlled limit (Riggs, A. D., Bourgeoise, S. & Cohn, M. (1970) J. Mol. Biol. 53, 401-417). A multi-step model proposes an initial diffusion-controlled association with nonspecific DNA sequences, followed by facilitated translocation to the operator, either by "sliding" and/or "direct transfer" mechanisms (Berg, O. G., Winter, R. B. & von Hippel, P. H. (1981) Biochemistry 20, 6929-6948). "Sliding" is well documented for several DNA-binding proteins. In contrast, the contribution of a "direct transfer" mechanism, which requires a protein able to simultaneously bind two regions of DNA, has not been critically evaluated. To assess the importance of a "direct transfer" mechanism to the sequence-specific binding of gene regulatory proteins, a comparative kinetic study of association rates of several Escherichia coli gene regulatory proteins was undertaken. The tetrameric Lac repressor (LacI) and dimeric Gal repressor (GalR) were used as models of bidentate and monodentate protein ligands for DNA, respectively. In addition, a deletion mutant of LacI (LacI{dollar}\sp{lcub}adi{rcub}{dollar}) that self-associates only to dimers was also analyzed. These proteins are members of the "Lac and Gal repressor" family of prokaryotic transcriptional regulators (Weickert, M. J. & Adhya, S. (1992) J. Biol. Chem. 267, 15869-15874) that share an overall primary sequence homology. Since their C-terminal, sugar-binding domains exhibit homology with the prokaryotic periplasmic sugar-binding proteins (proteins for which high-resolution crystal structures were available), the structural similarity of the LacI and GalR monomers was established through the construction of a homology-based molecular model of the GalR C-terminal domain. A novel time-resolved quench-flow quantitative DNase I "footprinting" method was developed in order to study the sequence-specific association kinetics of the repressors at protein concentrations sufficient to maintain their oligomerization states. These studies demonstrate that the direct transfer mechanism makes a significant contribution to the rate of association of bidentate proteins.;The quench-flow "footprinting" approach was also used to study the sequence-specific binding of the TATA-binding protein (TBP), a general transcription factor required by all three eukaryotic RNA polymerases. The binding of TBP to the adenovirus E4 promoter was studied as a function of protein concentration and temperature. The value determined for the second-order rate constant at pH 7.4, 100 mM KCl, 5 mM MgCl{dollar}\sb2{dollar}, 1 mM CaCl{dollar}\sb2{dollar}, 30 {dollar}\sp\circ{dollar}C ({dollar}k\sb{lcub}a{rcub}{dollar} = (5.2 {dollar}\pm{dollar} 0.5) {dollar}\times{dollar} 10{dollar}\sp5{dollar} M{dollar}\sp{lcub}-1{rcub}{dollar} sec{dollar}\sp{lcub}-1{rcub}{dollar}) is consistent with results obtained by gel mobility-shift and fluorescence anisotropy analysis (Hoopes, B. C., LeBlanc, J. F. & Hawley, D. K. (1992) J. Biol. Chem. 267, 11539-11547; Perez-Howard, G. M., Weil, T. & Beechem, J. M. (1995) Biochemistry 34, 8005-8017). The Arrhenius plot for TBP association is nonlinear, yielding a value of {dollar}\rm\Delta C\sb{lcub}p{rcub}\sp\circ{dollar} = {dollar}-{dollar}3.2 {dollar}\pm{dollar} 0.3 kcal/mol{dollar}\cdot{dollar}K. Congruence with the value determined from concurrent thermodynamic studies (Petri, V., Hsieh, M. & Brenowitz, M. (1995) Biochemistry 34, 9977-9984) suggests that {dollar}\rm\Delta C\sb{lcub}p{rcub}\sp\circ{dollar} of TBP-E4 promoter interaction is partitioned principally, if not entirely, in the rate-limiting step of the association reaction.