Direct imaging of phosphorylation-dependent conformational change and DNA binding of CREB by electron microscopy

Background: The second messenger cAMP stimulates the expression of numerous genes through the PKA-dependent phosphorylation of CREB. The cAMP-regulate d transcription factor CREB undergoes conformational change in response to phosphorylation by PKA at Ser 133. The phosphorylation enables interaction between the kinase-inducible domain (KID) of CREB and KIX domain of CREB bi nding protein (CBP). Results: To understand the activation mechanism of CREB-mediated gene expre ssion, we performed the electron-microscope imaging of the transcription ma chinery. We improved the metal shadowing techniques to achieve higher resol ution to detect phosphorylation-induced conformation change of the protein. Homodimer formation of CREB and the complex formation of phosphorylated CR EB with CBP were observed under the electron microscope. The binding of the CREB dimer to CREs on the somatostatin and tyrosine hydroxylase promoters were also visualized directly and stereoscopically. Conclusions: Greatly improved resolution achieved by our modified metal sha dowing techniques makes it possible to visualize that the shape of CREB hom odimer was changed in phosphorylation-dependent manner and that the promote r DNA strands containing CREs appeared to be bent and twisted slightly by t he holding in the crevice of the CREB homodimer. This method may be applica ble to visualize transcriptional activation process of nuclear receptors or general transcription machinery.