Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay.
We describe a new assay system that allows a rapid, direct, and quantitative detection of promoter-dependent in vitro transcription by RNA polymerase II. The template used is a hybrid plasmid containing the adenovirus major late promoter linked to a synthetic 400-base-pair DNA fragment that lacks cytidine residues on the transcribed strand--i.e., generates a transcript with no guanosine residues. In vitro transcriptions are carried out in the absence of GTP or, if the reactions contain GTP, in the presence of RNase T1 and the chain terminator 3'-0-methyl-GTP. Under these conditions the only RNAs that can accumulate, whether from a circular or linearized DNA template, are the 400-nucleotide RNase T1-resistant transcripts resulting from accurate initiation at the major late promoter. Thus, specific transcription can be directly monitored by conventional RNA quantitation methods. Using this fast assay, we show that three basic transcription factors, TFIIB, TFIID, and TFIIE, are absolutely required, in addition to the RNA polymerase II, for specific transcription initiation from the adenovirus major late promoter. Units of activity can be defined for each of these individual components. The applicability of this kind of assay to other systems is discussed.
D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D009876:Operon; D010957:Plasmids; D012319:RNA Polymerase II / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D006160:Guanosine Triphosphate; D012319:RNA Polymerase II; D006163:Ribonuclease T1