Probing the Effects of Highly Bent and Twisted DNA on Transcription by RNA Polymerase

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Cells contain a large number of genes that are expressed in complex patterns to ensure proper cellular function and differentiation. It is well established that regulatory proteins play a central role in transcriptional control and regulate the activity of RNA polymerases (RNAP). In addition, various lines of evidence related to supercoiling, looped DNA topologies, and chromatin remodeling suggest that RNAP activity may also be regulated via the nano-mechanical properties of DNA. We hypothesize that bending and twisting of the DNA template directly regulate the activity of RNAPs. To test this hypothesis we will generate DNA minicircles of a length of about 100 base pairs to introduce bending strain energy and torsion into DNA templates of a physiologically relevant magnitude in the absence of repressors or any other regulatory proteins. Using these minicircle DNA templates and molecular beacon-based transcription assays to directly detect synthesized mRNA, we will characterize changes in the kinetic properties of T7-RNAP. We will also develop assays to track the transcription from single DNA minicircles and to directly modulate torsion in DNA molecules while recording the activity of RNAP. Lastly, we will utilize computational rod models to predict the relationship between DNA topology and transcription. Our research on the role of DNA template mechanics on the activity of RNA polymerases has broader implications for the biological, medical and physical sciences. The work will close an important gap in our fundamental knowledge of the biology of DNA mechanics. The proposed assays will facilitate a detailed characterization of the effects that DNA template topologies and mechanics have on the activity of RNAPs. If successful, this research expands our understanding of transcriptional regulation and compels a paradigm shift in how we think about and approach genetic switches and gene regulation. Thus, the work has important implications for the medical sciences and will contribute to a rational understanding of cell regulation, cellular control and tissue engineering. The multidisciplinary approach and techniques developed here will also benefit the study of numerous other DNA-binding enzymes, other areas of gene regulation and the study of molecular motors and the cytoskeleton.