Multiple forms of the human gene-specific transcription factor USF. II. DNA binding properties and transcriptional activity of the purified HeLa USF.
The gene-specific upstream stimulatory transcription factor (USF) is required for maximal expression of the adenovirus major late promoter in vivo as well as in vitro. We have examined the DNA binding and transcriptional properties of USF purified to near-homogeneity from HeLa cell nuclei (Sawadogo, M., Van Dyke, M. W., Gregor, P. D., and Roeder, R. G. (1988) J. Biol. Chem. 263, 11985-11993). The 44-and 43,000-dalton forms of USF displayed identical affinities for the major late promoter upstream sequence. Specific binding parameters were greatly influenced by neighboring sequences, but not by the topological state of the DNA. The dissociation rate was highly dependent upon the concentration of competitor DNA, indicating that USF can efficiently transfer from one binding site to another by passing through a doubly bound intermediate state (direct transfer mechanism). Transcription stimulation by purified USF showed titration curves identical to those observed with cruder preparations of the transcription factor. However, the overall stimulation observed at saturating USF concentration was significantly lower with the purified protein. By contrast, interaction with TATA box-binding RNA polymerase II transcription factor D was observed with both USF-containing fractions. This could suggest the existence of two different mechanisms for upstream sequence-dependent transcription stimulation, where one critical component (or some necessary modification of the upstream factor itself) may be missing in reactions reconstituted with purified USF.
Sawadogo
M
M
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021-6399.
D000256:Adenoviridae / Q000235:genetics*; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000032:analysis; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008970:Molecular Weight; D010957:Plasmids; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D051720:Upstream Stimulatory Factors
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D035362:Transcription Factor TFIID; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; D015636:Magnesium Chloride; D011189:Potassium Chloride; D008274:Magnesium