Multiple forms of the human gene-specific transcription factor USF. I. Complete purification and identification of USF from HeLa cell nuclei.
The human gene-specific upstream stimulatory transcription factor (USF) is required, both in vivo and in vitro, for maximal expression of the major late promoter (MLP) of adenovirus. We report here the complete purification and identification of USF from HeLa cell nuclei. The protein was followed throughout its purification using a quantitative filter binding assay. With a combination of classical purification techniques and fast-flow protein liquid chromatography, USF can be purified to homogeneity starting either with a standard HeLa cell nuclear extract or with a higher salt extract from (lysed) HeLa cell nuclei (nuclear pellet extract). Approximately 20,000-fold purification from the nuclear pellet extract and 80,000-fold from the nuclear extract are necessary to obtain homogeneous preparations of the transcription factor. A maximum of 20,000 molecules of USF appear to be present in HeLa cells. Two major forms of the USF protein can be distinguished both by their slightly different mobilities in sodium dodecyl sulfate gel electrophoresis (apparent molecular weights 44,000 and 43,000, respectively) and by different electrophoretic mobilities of the corresponding protein-DNA complexes. Both forms of USF are heat-stable and interact with the MLP as monomers. Antibodies elicited against purified HeLa USF interact with the transcription factor bound to the MLP upstream element.
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Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021-6999.
Van Dyke
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Gregor
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Roeder
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D000256:Adenoviridae / Q000235:genetics; D002467:Cell Nucleus / Q000032:analysis*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005590:Fractional Precipitation; D006367:HeLa Cells / Q000032:analysis; D006358:Hot Temperature; D006801:Humans; D007118:Immunoassay; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D014157:Transcription Factors / Q000032:analysis* / Q000378:metabolism; D014158:Transcription, Genetic; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D010455:Peptides; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; D004247:DNA