Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements

Abstract

The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Critical for understanding how p53 regulates gene expression is unraveling the fundamental mechanisms by which it binds to REs. Toward this goal we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of tetrameric p53 to five native REs in real time under equilibrium conditions. We found little evidence of dimer/DNA complexes as intermediates to the formation or dissociation of p53 tetramer/DNA complexes; however, tetramer/DNA complexes can exchange dimers at some REs. Determining rate constants for association and dissociation revealed two kinetically distinguishable populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing the more favorable binding sequences form the least kinetically stable complexes. Together our real time measurements provide insight into mechanisms with which tetrameric p53 forms complexes on different native REs.