Reaction parameters of TFIIIA-induced supercoiling catalyzed by a Xenopus laevis cell-free extract.

AUTOR(ES)

Sekiguchi, J A

RESUMO

In addition to its fundamental role of nucleating the formation of stable transcription complexes, the Xenopus laevis 5S RNA specific transcription factor, TFIIIA, promotes a variety of DNA-associated metabolic reactions. We report that TFIIIA can induce a DNA supercoiling catalyzed by the Xenopus laevis S-150 cell-free extract on plasmids containing a single copy of the Xenopus 5S RNA gene (somatic-type). Stimulated supercoiling occurs in the presence of high concentrations of ATP (4 mM) and at a factor to DNA ratio of 1 through a mechanism most likely involving type I topoisomerase. The highest level of stimulated supercoiling occurs when TFIIIA is incubated with DNA prior to the addition of the S-150 extract. Taken together, the experiments outlined in this report establish a reliable and seminal system in which TFIIIA-induced DNA supercoiling can be observed reproducibly.

Documentos Relacionados

• Transport of vesicular stomatitis virus glycoprotein in a cell-free extract.
• Isolation of an active transcription initiation complex from HeLa cell-free extract.
• Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract.
• In vitro chromatin assembly promoted by the Xenopus laevis S-150 cell-free extract is enhanced by treatment with RNase A.
• Transcription of cloned transfer RNA genes from Drosophila melanogaster in a homologous cell-free extract.