Changes in DNA topology can modulate in vitro transcription of certain RNA polymerase III genes

The role of DNA supercoiling in eukaryotic gene expression is not fully understood. The objective of this study was to examine the regulation of in vitro chromatin assembly by topological alterations in the DNA template using a cell-free extract from Xenopus laevis oocytes (S-150). The results suggest that input DNA topology may be a determining factor in controlling the transcriptional activity of the Xenopus tRNA and one particular 5S gene. When the input topology is supercoiled, high levels of transcription are observed, whereas input relaxed DNA is transcribed to a much lower extent. Transcription from an input relaxed template is stimulated by the addition of supercoiled nonspecific, vector DNA. Furthermore, in direct competition experiments, supercoiled DNA molecules were shown to be transcriptionally dominant over relaxed DNA molecules. Taken together, these data suggest that the efficiency with which a repressor or activator binding protein interacts with DNA may be significantly influenced by the topological status of its target. We demonstrate that modulation of reaction parameters which alter the normal topological processing events catalyzed by the S-150 can dramatically influence the level of gene expression.