Probing Endogenous RNA Polymerase II Pre-initiation Complexes by Electrophoretic Mobility Shift Assay
RNA polymerase II (Pol II) plays a crucial role in eukaryotic biology since it is necessary for the expression of all protein-coding genes as well as most microRNAs and several small nuclear RNAs. Pol II is specifically recruited to core promoter DNA via its association with general transcription factors (GTFs) that possess DNA binding activity such as TFIID, TFIIA, and TFIIB. The large multi-protein assemblies of Pol II together with the GTFs required for productive transcription are termed pre-initiation complexes (PICs). To date, studies of the interaction of PICs with promoter DNA have relied on the use of purified or recombinant GTFs. Recent findings have demonstrated an astonishing diversity in the function of core promoters as well as in the protein composition of PICs. The currently known subset of GTFs alone cannot account for observed PIC and core promoter diversity. In order to identify the full complement of factors that impart PIC specificity, techniques to analyze the DNA binding of endogenous PIC are essential. Analysis of endogenous PIC formation has remained out of reach due to technical hurdles presumably including the large size of endogenous PIC, their highly dynamic association with core promoters, and the complex topology of DNA bound to PIC. We have optimized electrophoretic mobility shift assays (EMSAs) to achieve the detection of endogenous Pol II PIC from nuclear extracts of human cells. Here, we provide a robust and sensitive EMSA method for the analysis of endogenous Pol II PICs.
Key wordsPre-initiation complex RNA polymerase II TFIID TATA-binding protein Electrophoretic mobility shift assay TBP-associated factor General transcription factor
We thank Drs. Arndt Benecke and Antonio Conconi for critical comments on the manuscript. We are grateful to Dr. Laszlo Tora for the generous gift of monoclonal antibodies directed against TFIID subunits.