Reconstitution of recombinant TFIIH that can mediate activator-dependent transcription

Aya Fukuda, Jun Yamauchi, Shwu Yuan Wu, Cheng Ming Chiang, Masami Muramatsu, Koji Hisatake

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)707-719
Number of pages13
JournalGenes to Cells
Volume6
Issue number8
DOIs
StatePublished - 2001

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Transcriptional Activation
Baculoviridae
General Transcription Factors
DNA Helicases
Activation Analysis
RNA Polymerase II
HeLa Cells
Phosphotransferases
Adenosine Triphosphate
Viruses
Proteins
carboxy-terminal domain kinase

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Reconstitution of recombinant TFIIH that can mediate activator-dependent transcription. / Fukuda, Aya; Yamauchi, Jun; Wu, Shwu Yuan; Chiang, Cheng Ming; Muramatsu, Masami; Hisatake, Koji.

In: Genes to Cells, Vol. 6, No. 8, 2001, p. 707-719.

Research output: Contribution to journalArticle

Fukuda, Aya ; Yamauchi, Jun ; Wu, Shwu Yuan ; Chiang, Cheng Ming ; Muramatsu, Masami ; Hisatake, Koji. / Reconstitution of recombinant TFIIH that can mediate activator-dependent transcription. In: Genes to Cells. 2001 ; Vol. 6, No. 8. pp. 707-719.
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