Characterizing the Effects of Highly Bent DNA on Transcription

Abstract

It is well-established that many transcriptional repressors such as the loop-forming lactose or galactose repressors tightly bend DNA. However, it remains unclear whether this bending directly affects the activity of RNA polymerase. Characterizing the effects of repressor-bound, highly stressed DNA on transcription is complicated by the inability to decouple the mechanical state of the DNA template from the kinetics of repressor binding, which itself will be influenced by the energy required for the repressor to strain the DNA. To our knowledge there currently exists no established assay capable of quantifying transcription by RNA polymerase from highly bent DNA templates which mimic the bending induced by loop-forming repressors in the absence of other DNA-binding proteins. We have developed a fluorescence-based in vitro assay capable of addressing this experimental limitation by exploiting the ability of DNA minicircles to impose both varying degrees of bending and twist. We are now applying this assay to characterize the rates of transcription by T7 RNA polymerase from highly stressed templates. We hypothesize that the activity of T7 RNA polymerase will be reduced on templates that are bent with degrees of curvature comparable to the loops generated by loop-forming repressor proteins. Contrary to our initial hypothesis, preliminary data suggests that at least one such highly bent DNA template is in fact readily transcribed by T7 RNA polymerase. However, the degree to which the minicircle templates are twisted beyond the torsionally relaxed helical structure may play a greater-than-expected role on the activity of RNA polymerase, and must therefore be addressed. Conclusions resulting from these measurements will shed light on the mechanism by which loop-forming repressors affect transcription, and could reveal a more significant role for the mechanical state of the DNA than was previously thought.