Topoisomerase I enhances TFIID-TFIIA complex assembly during activation of transcription.
The mechanism of coactivation by DNA topoisomerase I (topo I) was examined in a highly defined in vitro transcription system containing Pol II and purified factors. Both stimulation of the basal reaction and coactivation occurred dependent on TAF(II)s. Activation was first observed at the TFIID-TFIIA stage of initiation and maximal activation required the concomitant presence of TFIID, TFIIA, topo I, and activator. Electrophoretic mobility shift assay demonstrated a dramatic enhancement in the formation of the TFIID-TFIIA complex by topo I and activator, dependent on the TAF(II)s. DNase I footprinting confirmed this recruitment. A catalytically inactive topo I, which coactivated transcription, similarly stimulated the rapid formation of the TFIID-TFIIA complex in the presence of activator. A camptothecin-mediated DNA cleavage assay demonstrated the recruitment of topo I to the template by TFIID. Topo I likely functions during activation by enhancing the formation of an active TFIID-TFIIA complex on the promoter.
Shykind
B M
BM
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139-4307, USA.
Kim
J
J
Stewart
L
L
Champoux
J J
JJ
Sharp
P A
PA
D002166:Camptothecin; D018983:DNA Footprinting; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000378:metabolism; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism*; D015533:Transcriptional Activation
D005656:Fungal Proteins; C070633:Gal-VP16; D015534:Trans-Activators; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin