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- Title
Reconstitution of recombinant TFIIH that can mediate activator-dependent transcription.
- Authors
Fukuda, Aya; Yamauchi, Jun; Wu, Shwu-Yuan; Chiang, Cheng-Ming; Muramatsu, Masami; Hisatake, Koji
- Abstract
Background TFIIH is one of the general transcription factors required for accurate transcription of protein-coding genes by RNA polymerase II. TFIIH has helicase and kinase activities, plays a role in promoter opening and promoter escape, and is also implicated in efficient activator-dependent transcription. Results We have established a reconstitution system of recombinant TFIIH using a three-virus baculovirus expression system. The recombinant TFIIH was active in CTD kinase and DNA helicase assays, and showed both basal and activator-dependent transcriptional activities that were indistinguishable from those of HeLa cell-derived TFIIH. Further analyses using recombinant TFIIH confirmed a critical role of TFIIH in activator-dependent transcription. The dose response of TFIIH in activator-dependent transcription suggested that mere recruitment of TFIIH is not sufficient for transcriptional activation. The sensitivity of activator-dependent transcription to nonhydrolysable ATP analogues indicated the importance of the enzymatic activities of TFIIH in transcriptional activation. Conclusions Our results raise a possibility that transcriptional activation by GAL4-VP16 requires enzymatic activities. Recombinant TFIIH reconstituted from this baculovirus system should be useful for analysis of the mechanisms of activation by GAL4-VP16.
- Subjects
BACULOVIRUSES; GENETIC transcription; ADENOSINE triphosphate
- Publication
Genes to Cells, 2001, Vol 6, Issue 8, p707
- ISSN
1356-9597
- Publication type
Article
- DOI
10.1046/j.1365-2443.2001.00456.x