Supercoil-induced unusual DNA structures as transcriptional block

Abstract

The transcriptional activity of pBR322 form V DNA template, a topologically unlinked, highly supercoiled molecule having unusual structures around or within coding regions was studied. Significant transcription was observed in vitro from this template despite high levels of supercoiling. An attenuated transcript, initiated accurately from the P4 promoter of rep gene, was observed which indicated pausing of E. coli RNA polymerase within the gene. This pausing could be removed by relieving the torsional stress implying that a supercoil induced structural alteration within the gene was acting as a transcriptional block. A stabilized unusual structure, most likely a cruciform, was found to be responsible for the elongation block. Absence of initiation from the tet^R gene was correlated with the unusual structure present within its promoter region in form V DNA. These in vitro studies show that structural alterations within natural DNA could act as transcriptional blocks both at the level of initiation and elongation.