Reaction parameters of TFIIIA-induced supercoiling catalyzed by a Xenopus laevis cell-free extract.
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.
Sekiguchi
J A
JA
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Kmiec
E B
EB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002474:Cell-Free System; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D006593:Hexokinase / Q000378:metabolism; D007700:Kinetics; D010957:Plasmids; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000378:metabolism*; D014982:Xenopus laevis
D004275:DNA, Ribosomal; D004278:DNA, Superhelical; D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D006593:Hexokinase