TATA box-mediated in vitro transcription by RNA polymerase III. Evidence for TATA-binding protein in a polymerase III type complex.
We have shown previously that under specific conditions, a TATA box will mediate efficient in vitro transcription by RNA polymerase (pol) III in the absence of a PSE or other promoter elements. The reaction requires a HeLa cell phosphocellulose protein fraction, fraction B, which must be preincubated with the template DNA. Fraction B does not contain any detectable pol II type transcription factor IID (TFIID) activity. In this report, the relationship between fraction B and TFIID was further examined. Purified human TATA-binding protein (TBP) can substitute for fraction B to mediate TATA-dependent pol III transcription. Both TBP and fraction B prefer a reverse TATA box for pol III transcription, yet TBP bound to a reverse TATA box functions poorly for pol II transcription. Like TFIID, fraction B forms a template-committed complex with TATA-containing promoters. TBP, however, will not template commit for pol III transcription unless premixed with phosphocellulose fraction C. TBP-mediated pol III transcription is also more sensitive to the detergent Sarkosyl (N-lauroylsarcosine, Sigma) than is the fraction B reaction unless it is premixed with fraction C. Together, the data suggest that TBP can complex with a component of fraction C, and this complex is then functionally equivalent to fraction B. We propose that fraction B contains TBP in a complex with some other component(s) of the pol III transcription machinery and that this B complex TBP may be specific for pol III transcription.
Mitchell
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Du Pont Merck Pharmaceutical Co., Wilmington, Delaware 19880-0328.
Benfield
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PA
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003902:Detergents; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D012320:RNA Polymerase III / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012521:Sarcosine / Q000031:analogs & derivatives; D016385:TATA Box; D035181:TATA-Box Binding Protein; D013698:Templates, Genetic; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D003902:Detergents; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; C025231:sarkosyl; D012319:RNA Polymerase II; D012320:RNA Polymerase III; D012521:Sarcosine