Characterizing the Relationship Between DNA Bending and Transcription Elongation By T7 RNA Polymerase

It is well recognized that transcriptional repression is mediated by the binding of a repressor protein to the DNA template. Some repressors, such as the lactose repressor (LacR), bind two sites on the DNA, forcing the DNA into a tight loop which includes the promoter. Unlike other repression mechanisms such as steric blocking, RNA polymerase (RNAP) is not physically prevented from binding to the promoter in these loops; in fact, it binds with greater affinity. Furthermore, it has been shown that LacR-induced repression depends directly on the loop size - as the loop becomes larger, repression decreases although LacR binds with higher affinity. This apparent contradiction can be resolved by considering the mechanical stress imparted on the DNA: in this case, we hypothesize that as loops increase in size, LacR repression decreases because the loop itself becomes more flexible. In order to elucidate the potential role of mechanical stress in transcriptional regulation, we have developed an assay capable of measuring transcription from DNA minicircles sustaining various levels of bending stress comparable to repressor loops. Using fluorescently labeled molecular beacons capable of hybridizing to a predefined portion of the transcript we have been able to measure the transcriptional elongation rate of bacteriophage T7 RNAP. We hypothesize that, in the absence of regulatory proteins, bending stresses are sufficient to repress transcription. Indeed, preliminary data confirm that tightly looped DNA can inhibit T7RNAP elongation, and we are currently expanding the analysis to minicircles with different degrees of bending to fully explore this relationship. Our study establishes for the first time that DNA bending is sufficient to repress transcription and necessitates the consideration of template mechanics in other transcription systems particularly those involving repressors known to significantly deform DNA tertiary structure.