Transcription by Escherichia coli RNA polymerase (RNAP), a well-characterized member of the multisubunit RNAP family, involves several mechanistic steps inaccessible to methods that study static structures or molecular ensembles. To understand transcription mechanisms, it is necessary to uncover and analyze dynamic, transient, and non-equilibrium steps along the transcription pathway. Single-molecule detection (SMD) is a new set of tools that can stand up to this challenge by monitoring the real-time behavior of individual transcription complexes. We have developed single-molecule Fluorescence Resonance Energy Transfer (smFRET) combined with alternating-laser excitation in order to study the structure and dynamics of transcription complexes. We propose to use this method to understand transcription by analyzing poorly-characterized transitions in transcription complexes; several of these transitions are extremely important for transcriptional regulation, since they form the steps where transcription factors control gene expression. We propose to focus on multistep transitions: the transitions occurring on the path from RNA polymerase to the formation of RNA polymerase-promoter open complex, the transitions occurring on the path from RNA polymerase-promoter open complex to initial transcribing complexes, and transitions occurring on the path from initial transcribing complexes to a mature elongation complex. The results of the proposed work will allow direct observation of structural and mechanistic heterogeneity of transcription complexes; validate or disprove models proposed after decades of genetic, biochemical, and structural analysis of transcription that were not validated experimentally; and will allow generation of real-time, molecular """"""""movies"""""""" of individual, functional RNAP molecules operating on DNA. The high homology of E. coli RNAP polymerase with its eukaryotic counterparts ensures that mechanistic insights obtained from the proposed work will be directly extrapolated to eukaryotic transcription and will greatly enhance understanding of transcription-associated human diseases, such as various forms of cancer, (since numerous oncogenes and tumor-suppressor genes are transcription factors), developmental defects, and other pathological conditions. The proposed methods are applicable to the analysis of nucleoprotein complexes present in DNA replication, DNA recombination, DNA repair, RNA processing and RNA translation, and when combined with advances in site-specific labeling, will allow the study of such processes in living cells.
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