In vitro transcription from Bacillus subtilis nucleoids by homologous RNA polymerase

The effect of DNA structural features on RNA synthesis was investigated. Purified Bacillus subtilis nucleoids templated from vegetative cells were transcribed by the homologous RNA polymerase using Hg-UTP as one of the nucleotide substrates. Low RNA polymerase/DNA ratios were used during transcription in order to avoid nonspecific initiations. The rate of synthesis of total RNA was 40% greater on nucleoid templates than on naked DNA. The proportion of asymmetric transcript synthesized on nucleoid templates (HvsL strand transcripts) was close to that observed in vivo, whereas with naked DNA this value was at least 3-times lower. The percentage of rRNA relative to the total RNA, synthesized in the in vitro system with the nucleoid template, approaches the rate of the in vivo transcription. The size of the RNA synthesized on nucleoids was large and heterogeneous while with naked DNA it was homogeneous and of about 6 S. Our results suggest that the supercoiled, folded nucleoids may retain some of the structural features responsible for the regulation of RNA synthesis in vivo.