Identification and purification of EBP1: a HeLa cell protein that binds to a region overlapping the 'core' of the SV40 enhancer.
The SV40 enhancer consists of multiple DNA sequence motifs that are recognized by a variety of trans-acting factors. Using DNase I protection and a gel electrophoresis DNA-binding assay, we identified a HeLa cell protein (EBP1) that binds to the 'core' region of the SV40 enhancer. A short double-stranded synthetic oligonucleotide containing the binding site for EBP1 was used to assay for EBP1 activity and to purify a 57,000-m.w. polypeptide by recognition site affinity chromatography. Bromodeoxyuracil cross-linking identified a 60,000-m.w. species as the polypeptide responsible for the DNA-binding activity. Analysis of the DNA sequences required for EBP1 binding indicated that EBP1 could be distinguished from a number of recently characterized proteins (EBP20, AP-2, and AP-3) by its binding to a variety of mutant templates. Correlation of the in vivo transcriptional activity of wild-type and mutated enhancers with EBP1 binding indicates that this protein may be important for SV40 enhancer activity because mutations that abolish EBP1 binding also have a severe deleterious effect on transcription.
Clark
L
L
Department of Biochemistry and Microbiology, University of St. Andrews, Fife, Scotland.
Pollock
R M
RM
Hay
R T
RT
D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013539:Simian virus 40 / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic
D004279:DNA, Viral; D014157:Transcription Factors