Kinetic and Conformational Characterization of Transcriptional Activator-Coactivator Interactions.

[摘要] Kinetic and Conformational Characterization of Transcriptional Activator-Coactivator InteractionsInitiation of transcription is achieved through a series of coupled binding equilibria commenced by interactions between DNA-bound transcriptional activators and coactivators. There is great need to understand the mechanism of these activator-coactivator interactions and design artificial transcriptional regulators as probes or potential therapeutics. However, the key mechanistic features responsible for the differential transcriptional output of these activators are yet to be well-defined. The focus of this dissertation work has been to dissect the kinetic and structural characteristics of transcriptional activator-coactivator interactions and examine the effects of small molecule modulators on these interactions.Utilizing fluorescence stopped-flow, we measured the transient-state kinetics of the transcriptional activation domains (TADs) of the activators Gal4, Gcn4 and VP16 in their DNA-bound forms binding to the coactivator Med15. We determined that they interact through the same two-step binding mechanism: an initial rapid bimolecular association step followed by a slower conformational change step. Additional analysis suggests that the tendency for an activator to undergo conformational change correlates with both its overall affinity to the coactivator and its transcriptional activity in vivo.This mechanistic study of activator-coactivator interactions was further applied to the more conformationally defined system of TADs (MLL and pKID) binding cooperatively to the coactivator KIX. The study showed that both TADs bind to KIX through a two-step mechanism similar to that of TADs binding to Med15. A small molecule fragment 1-10 from a Tethering screen covalently tethers to a cysteine mutant of the coactivator KIX domain of CBP at the MLL binding site. The additional stabilizing effect of 1-10 tethering to KIX enabled me to obtain a crystal structure of 1-10—KIX L664C. Additionally, I found that 1-10 elicits varying allosteric effects on the opposite pKID binding site of KIX, depending on the site at which it tethers. I used 1-10 tethered at different cysteine mutations as well as the MLL peptide as probes to study the allosteric effects of KIX’s pKID binding through transient-state kinetics. The results suggest that the dissociation rate constant koff between pKID and KIX correlate with their overall binding affinity KD.

[发布机构] University of Michigan

[效力级别] Biological Chemistry

[关键词] Transcriptional Activator-coactivator Interactions;Biological Chemistry;Science;Chemical Biology