Factors involved in specific transcription by mammalian RNA polymerase II: purification and characterization of general transcription factor TFIIE.
Human transcription factor TFIIE, a ubiquitous factor required for transcription initiation by RNA polymerase II, was purified to homogeneity by a combination of conventional and HPLC steps. The purified TFIIE contained equimolar amounts of 57-kDa (TFIIE-alpha) and 34-kDa (TFIIE-beta) polypeptides that were judged to be functional subunits on the basis of their copurification with transcriptional activity and the recovery of activity following renaturation of polypeptides separated by reverse-phase HPLC. TFIIE-alpha had an independent TFIIE activity whereas TFIIE-beta had no activity alone but enhanced the activity of TFIIE-alpha. In conjunction with gel filtration studies, which indicated a molecular mass of approximately 180 kDa for the native protein, these results suggested that TFIIE is a heterotetramer containing two alpha and two beta polypeptides. Functional studies with the purified TFIIE demonstrated that it is a general initiation factor, required for all of the genes tested, but it failed to show any DNA-dependent ATPase activity.
Ohkuma
Y
Y
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, NY 10021.
Sumimoto
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H
Horikoshi
M
M
Roeder
R G
RG
D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012319:RNA Polymerase II / Q000378:metabolism*; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D012319:RNA Polymerase II