Investigating mechanisms of RNA polymerase II transcription and regulation using single molecule fluorescence

Expression of genes encoded in DNA starts with transcription – the process whereby one strand of DNA is copied to make RNA. Transcription requires that many different proteins and the DNA assemble into large complexes to enable accurate and timely gene expression in appropriate cellular contexts. This project will study the assembly and disassembly of key complexes involved in the early stages of transcription using microscopy techniques that allow observation of single molecules. Monitoring individual molecules will reveal new insights into diverse behaviors of proteins and DNA that are important for controlling gene expression. This project will also have broad educational impacts through training of graduate and undergraduate students and the development of a new laboratory curriculum. The specific objectives of the project are to investigate the macromolecular interactions that govern binding of architectural proteins and transcription factors to nucleosomes, and the molecular transformations that occur in RNA polymerase II (Pol II) transcription complexes. The experiments employ in vitro single molecule fluorescence techniques, which are ideally suited to study rapid on/off interactions, molecular scanning, and heterogeneity in the systems under investigation, thus providing deep insights into function. One objective is to investigate how the human architectural protein HMGB1 is able to rapidly traverse nucleosomes and aid transcription factor p53 in binding its recognition site. The second objective is to resolve how the binding/dissociation of general transcription factor TFIIE to Pol II complexes controls transcriptional activity. Unique to this study, active and inactive complexes will be distinguished, thereby directly relating TFIIE behavior to transcriptional activity. The knowledge gained from this research will stimulate new models that will increase understanding of transcriptional regulation. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.