Local domains of supercoiling activate a eukaryotic promoter in vivo

EXPERIMENTS correlating template topology with transcriptional activity suggest that DNA topology plays a role in eukaryotic gene expression1–4. Linear templates transfected into cultured cells produce far fewer transcripts than do circular transcription templates3, and no transcripts can be detected from linear templates injected into Xenopus oocytes1,2. Further, when transcriptionally active circular templates in Xenopus oocytes are linearized by injection of a restriction enzyme, transcription dramatically decreases. Here we show that transcription by phage T7 RNA polymerase from a divergent promoter can partially replace the requirement for circular Xenopus ribosomal RNA transcription templates in Xenopus oocytes. Supercoiled domains can apparently be generated on short pieces of DNA having no known sequences that result in association with the nuclear architecture, suggesting that localized, transient domains of supercoiling fulfil the minimum topological needs for Xenopus rRNA transcription

[1]  R. Hertzberg,et al.  Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. , 1985, The Journal of biological chemistry.

[2]  R. Wolff,et al.  Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes , 1986, Molecular and cellular biology.

[3]  P. Labhart,et al.  Xenopus ribosomal gene enhancers function when inserted inside the gene they enhance. , 1985, Nucleic acids research.

[4]  H. Weintraub,et al.  Expression of transfected DNA depends on DNA topology , 1986, Cell.

[5]  Y. Tsao,et al.  Transcription-driven supercoiling of DNA: Direct biochemical evidence from in vitro studies , 1989, Cell.

[6]  J. R. Paulson,et al.  The structure of histone-depleted metaphase chromosomes , 1977, Cell.

[7]  G. Giaever,et al.  Supercoiling of intracellular DNA can occur in eukaryotic cells , 1988, Cell.

[8]  J. Wang,et al.  Supercoiling of the DNA template during transcription. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[9]  P. Hanawalt,et al.  Localized torsional tension in the DNA of human cells. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[10]  M. Dunaway.  A transcription factor, TFIS, interacts with both the promoter and enhancer of the Xenopus rRNA genes. , 1989, Genes & development.

[11]  P. Labhart,et al.  Enhancer-like properties of the 60/81 bp elements in the ribosomal gene spacer of Xenopus laevis , 1984, Cell.

[12]  R. Reeder,et al.  Effect of topological constraint on transcription of ribosomal DNA in Xenopus oocytes. Comparison of plasmid and endogenous genes. , 1984, Journal of molecular biology.

[13]  M. Dunaway,et al.  Transactivation of the Xenopus rRNA gene promoter by its enhancer , 1989, Nature.

[14]  H. Weintraub,et al.  Transcription of DNA injected into Xenopus oocytes is influenced by template topology , 1983, Nature.

[15]  Charles Weissmann,et al.  Unusual splice sites revealed by mutagenic inactivation of an authentic splice site of the rabbit β-globin gene , 1983, Nature.

[16]  S. Bell,et al.  The Xenopus ribosomal gene enhancers bind an essential polymerase I transcription factor, xUBF. , 1989, Genes & development.

[17]  J. Wang,et al.  Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase. , 1990, Science.

[18]  Q. Ju,et al.  Topoisomerases and yeast rRNA transcription: negative supercoiling stimulates initiation and topoisomerase activity is required for elongation. , 1992, Genes & development.

[19]  U. K. Laemmli,et al.  Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold , 1984, Cell.