Journul @‘Chromutogruphy, 5 10 (1990) 243-250 Elsevier Science Publishers B.V., Amsterdam CHROM. 22 424 Purification of the upstream element factor of the Adenovirus-2 major late promoter from HeLa and yeast by sequence-specific DNA affinity chromatography VINCENT MONCOLLIN, MATTHIEU GERARD and JEAN-MARC EGLY* Lahoratoire de GPnGtique MolPculaire des Eucaryotes du CNRS, Unit6 184 de Biologic Molkulaire et de GPnie GPn&ique de I’INSERM. Insfitut de Chimie Biologique. Facult6 de M6decine. II Rue Humann, 67085 Strusbourg C&de-x (France) ABSTRACT The purification to homogeneity of the Adenovirus-2 major late promoter (MLP) upstream element factor (UEF), a sequence specific transcription factor, which binds to upstream elements of various class B (II) genes, is reported. The protein was purified from HeLa cells and also from the yeast zyxwvutsrqponmlkjihgfedcba Saccharomyces cere- visiae, by using sequence-specific DNA affinity chromatography. The human (UEFh, 45 000 dalton) and the yeast (UEFy, 60 000 dalton) proteins protect the same se- quences over the MLP-IVa2 intergenic region: the MLP-UE (from nucleotide - 49 to - 67) and the IVa2-UE (from nucleotide - 98 to - 122 relative to the MLP initiation site). Both proteins have a higher affinity for the MLP-UE than for the IVa2-UE. INTRODUCTION Transcription of protein coding genes requires the presence of distinct promo- ter elements in the DNA template: (1) the initiation site and the TATA-box, a highly conserved AT-rich region located 30 base pairs (bp) and 40-l 10 bp upstream of the transcription start site in higher and lower eukaryotes, respectively, and (2) the up- stream elements which are located in the - 40 to - 110 region upstream from the start site (for a review, see ref. 1). Zn vivo and in vitro studies have shown that these promoter elements are the target of various transcription factors. While the TATA- box interacts with the TATA-box recognizing factor BTF12, also called TFIID3, both the upstream elements and enhancers bind sequence specific factors (e.g., Spl, HSTF, CBP) which have some regulatory functionI. The HeLa upstream element factor (UEF) of the Adenovirus-Zmajor late pro- moter (Ad2 MLP) had been shown to stimulate Ad2 MLP transcription both in vivo and in vitro through the binding at the MLP upstream element (MPL-UE located between positions - 49 and - 67 of the Ad2 MLP)4. Further it has also been suggest- ed that stimulation of transcription by the sequence-specific upstream factors occurs 0021-9673/90/$03.50 0 1990 Elsevier Science Publishers B.V.