Transcription of highly expressed bacterial genes occurs in stochastic bursts. Chong et al. now show that DNA gyrase regulates this transcriptional burst by decreasing positive DNA supercoiling. The authors designed an in vitro assay that enables transcription on individual chromosomal DNA loops to be monitored in real time and found that, during multiple rounds of transcription, supercoiling accumulated on the DNA template ahead of the RNA polymerase (known as positive supercoiling) and blocked transcription initiation and elongation. Notably, transcription initiation was restored following the addition of gyrase, which is the enzyme that resolves positive supercoiling. By using gyrase inhibitors or by overexpressing gyrase in Escherichia coli, the authors confirmed that this enzyme is the main regulator of transcriptional bursting. Future studies should clarify whether gyrase activity also regulates transcriptional bursting in eukaryotes.