SUNY at Albany
June 19-23, 2001
Energetics of the Sequence-Specific Interactions of the ETS Domain of Murine PU.1.
The DNA-binding domains of the ets family of transcription factors recognize purine-rich sequences containing the consensus sequence 5'-GGAA/T-3'. We are studying the energetics of the sequence-specific binding of the ets domain of murine PU.1 to the lB site of the Igl2-4 enhancer by gel-mobility shift, nitrocellulose filter binding, and hydroxyl radical foot-printing. Over the temperature range of 0 to 50ºC, the dissociation equilibrium constant, KD, is essentially independent of temperature. The magnitude of KD is in the nM range and it varies less than 10-fold over these temperatures, implying that the enthalpy of interaction DH ~ ?1 kcal mol-1. Within exper-imental error, the log KD decreases linearly with inverse temperature. In comparison to other DNA-protein interactions, this behaviour is unusual. For other systems, e.g., EcoRI, lac repressor, plots of log KD vs T-1 exhibit significant negative curvature, this indicates that a large nega-tive change in heat capacity DCp accompanies protein binding. We believe this to be the first example of a sequence-specific protein-DNA interaction whose energetic profile is more characteristic of nonspecific binding (i.e., small DH contribution, DCp ~ 0). The very minimal temperature dependence of DH was observed by mobility shift and nitro-cellulose filter binding. The salt dependence of DH has been investigated using mobility shift, at 150 mM and 250 mM Na+ there is no significant difference in the behaviour. Qualitative high-resolution footprinting of the interaction shows that there are no major changes in the contact surface of the protein and DNA as the conditions are changed.
Gregory Man Kai Poon and Robert B. Macgregor, Jr.
Department of Pharmaceutical Sciences
University of Toronto
19 Russell Street
Toronto, Ontario M5S 2S2 Canada