Tightly Looped DNA Represses Transcription Initiation by T7 RNA Polymerase

Abstract

DNA looping by gene repressors has long been recognized as a widely conserved paradigm for prokaryotic repression. Yet, the extent to which the mechanics of the looped DNA itself plays a direct, physical role in repressing RNA polymerase (RNAP) remains debated. Recently, we have shown that transcription elongation by the bacteriophage T7 RNAP (T7RNAP) is significantly repressed from tightly looped DNA templates (Biophys.J.99(4),2010). However, it remains unclear if mechanically stressed DNA templates also affect RNAP activity in other phases of transcription. During transcription initiation, a polymerase bound to a looped DNA template encounters a fundamentally mechanical challenge: it must transcribe the first ∼8-10 nucleotides of the template while maintaining contacts with the upstream promoter. Consequently, the polymerase must overtwist the torsionally constrained DNA template by nearly a full turn before it releases the promoter. We hypothesize that the mechanical properties of tightly looped DNA alone can be repressive to this process. To test this hypothesis, we have developed an assay that is capable of monitoring transcription initiation from DNA minicircle templates that sustain well-characterized amounts of bending and torsional stresses. Preliminary data suggests a substantial increase in the formation of abortive products from a 100-bp DNA minicircle compared to its linear counterpart. We interpret this observation to confirm our broad hypothesis that template mechanics can directly repress initial transcription. To determine if the observed repression is owed to the sharp bending curvature of the looped template or the torsional properties of the minicircle, we have generated minicircle templates that are untwisted, overtwisted, and nicked (and hence sustain no torsional stress). The ongoing characterization of these minicircle templates is expected to shed light on the molecular mechanism through which mechanical stresses in DNA templates can directly repress transcription initiation by T7RNAP.