Traditionally, responsive promoter sequences on DNA have been considered only passive docking sites for a pletora of DNA-binding proteins supposed to play the active hard role of gene expression. Several proteins have been pulled-down according to their ability to bind DNA sequences (i.e., far western blotting) and lot of plasmids were generated carrying any responsive DNA element upstream of a reporter gene to mainly study the activity of such proteins (i.e., transcription factors) and eventually discover new drugs. In other words, reporter assay data have been mainly queried to address gene expression from the protein stand-point (trans-action).
It is worth to note, that protein activity is longly known to be allosterically regulated by the binding of ligands or cofactors outside the protein’s active site. Now, Sebastian Meijsing and colleagues from the Yamamoto Lab, are shifting the balance toward cis-acting factors (the DNA itself). They propose in a Science report that DNA is a sequence-specific allosteric ligand for the nuclear receptor GR (glucocorticoid receptor). GR may be considered a ligand-activated transcription factor (i.e., it is activated by cortisol or dexametasone). The Yamamoto group exploited a classic luciferase assay to test the activity resulting from the interaction between GR (protein) on its GREs (DNA) during dexametasone stimulation.
|Molecular gymnast by:Elio Abbondanzieri|
Interestingly, odd differences were found in their elegant 2x2 experimental scheme in which either single-poing mutations on the receptor or on the responsive elements were combinatorially tested to finally postulate that DNA topology actively directs transcription similarly to an allosteric regulator. Gene expression , crystallographic, gel-shift and ChIP assays corroborate this intuition. Pay attention to your plasmids: DNA is more than a passive docking site.
Meijsing, S., Pufall, M., So, A., Bates, D., Chen, L., & Yamamoto, K. (2009). DNA Binding Site Sequence Directs Glucocorticoid Receptor Structure and Activity Science, 324 (5925), 407-410 DOI: 10.1126/science.1164265