Reconstitution of recombinant TFIIH that can mediate activator-dependent transcription.
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.
Fukuda
A
A
Department of Biochemistry, Saitama Medical School, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan.
Yamauchi
J
J
Wu
S Y
SY
Chiang
C M
CM
Muramatsu
M
M
Hisatake
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D016367:Baculoviridae; D003001:Cloning, Molecular / Q000379:methods; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C070633:Gal-VP16; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D012319:RNA Polymerase II; D004265:DNA Helicases