A downstream-element-binding factor facilitates assembly of a functional preinitiation complex at the simian virus 40 major late promoter.
Recent work has shown that many promoters recognized by eucaryotic RNA polymerase II contain essential sequences located downstream of the transcriptional initiation site. We show here that the activity of a promoter element centered 28 base pairs downstream of the simian virus 40 major late initiation site appears to be mediated by a DNA-binding protein, which was isolated by affinity chromatography from HeLa cell nuclear extracts. In the absence of the other components of the transcriptional machinery, the protein bound specifically but weakly to its recognition sequence, with a Kd of approximately 10(-8) M. Analysis of kinetic data showed that mutation of the downstream element decreased the number of functional preinitiation complexes assembled at the promoter without significantly altering the time required for half the complexes to assemble. This suggests that in the absence of the downstream activating protein, preinitiation complexes are at least partially assembled but are not transcriptionally competent.
Ayer
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DE
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Dynan
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WS
D001483:Base Sequence; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D012319:RNA Polymerase II; D003850:Deoxyribonuclease I