Integration of Protein Kinases into Transcription Complexes: Identifying Components of Immobilised In Vitro Pre-initiation Complexes

Regulation of gene expression is essential for coordinated cell growth and development. The de-regulation of certain genes is also recognised to contribute to both heritable and acquired disease. Transcription factors influence the assembly and activity of transcription complexes, which they achieve in part by recruiting co-activators to gene promoters to participate in the dynamic cycle of polymerase binding, initiation and escape from the promoter. Co-activator recruitment and accompanying post-translational modifications to components of promoter complexes appear to differ between genes and as a consequence of varying signal input. Thus a full understanding of transcriptional initiation and control will ultimately require the elucidation of these processes. The method described here was designed to detect the presence of proteins and post-translational modifications in complexes formed in vitro on gene-specific promoters. It has been used, among other things, to detect the recruitment of the Mitogen-Activated Protein (MAP) kinases ERK1 and ERK2 to the promoters of mitogen-responsive genes.