Stimulation of the adenovirus major late promoter in vitro by transcription factor USF is enhanced by the adenovirus DNA binding protein.
Previous studies have shown that the sequence-independent adenovirus DNA binding protein (DBP) increases transcription from several promoters, notably from the adenovirus major late promoter (MLP) and the adeno-associated virus P5 promoter, both of which contain a USF/MLTF binding site. In order to study this mechanism, we have investigated the effects of DBP on the binding of USF/MLTF to MLP and on transcription from MLP by a reconstituted in vitro system. As shown by gel retardation and DNase I footprinting, upon saturation of DNA, DBP enhances the binding affinity of USF43 to the promoter three- to fourfold without changing the footprint pattern. In contrast, the binding of the TATA box binding protein to the promoter is not influenced by DBP. No protein-protein interactions between DBP and USF43 could be observed in the absence of DNA, suggesting that enhanced binding is caused by a change in DNA structure induced by the DBP-DNA complex. Employing a transcription system reconstituted with purified general transcription factors, we show that USF43 enhances basal transcription and that USF43-dependent transcription is further increased by DBP, while DBP alone does not have an effect on basal transcription. Our results suggest that transcription enhancement by DBP is based on a specific increase in the binding of a transcription factor to a promoter through subtle changes in DNA structure, similar to the mechanism by which DBP stimulates the initiation of DNA replication.
Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
D C
DC
Zijderveld
F
F
d'Adda di Fagagna
M
M
Giacca
H T
HT
Timmers
P C
PC
van der Vliet
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051839:NFI Transcription Factors; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016385:TATA Box; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C080333:transcription factor nuclear factor 1; D003850:Deoxyribonuclease I