Optimization of a versatile in vitro transcription assay for the expression of multiple start site TATA-less promoters.
Previous work from our laboratory has allowed for the subdivision of RNA polymerase II TATA-less promoters into two classes: those that initiate at a single start site (SSS) and those that initiate at multiple start sites (MSS). MSS promoters are defined by the lack of a TATA box and the presence of a transcription initiation window and a downstream MED-1 element (GCTCCC/G) [Ince, T. A., and Scotto, K. W. (1995) J. Biol. Chem. 270, 30249-30252]. Further insight into the mechanisms regulating TATA-less MSS promoters has been hampered by the lack of an in vitro transcription assay in which multiple start sites can be reproduced. In the present study, we describe the development of a versatile in vitro transcription system optimized for the expression of MSS promoters, termed the multiple promoter comparison (MPC) assay. By alteration of assay parameters including template length, cation and nucleotide concentrations, and RNA isolation method, the accurate and robust transcription of two MSS promoters, pgp1 (hamster P-glycoprotein class I homologue) and HPRT (human hypoxanthine phosphoribosyltransferase), was accomplished. Moreover, both TATA-containing and TATA-less single start site promoters were also transcribed in the MPC assay, making this the first general in vitro transcription system for the simultaneous analysis of all three classes of RNA polymerase II genes.
Lin
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Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
Ince
T A
TA
Scotto
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KW
D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012319:RNA Polymerase II / Q000235:genetics*; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D011189:Potassium Chloride; D004247:DNA; D008156:Luciferases; D012319:RNA Polymerase II; D008274:Magnesium