Protein-DNA recognition is often mediated by a small domain containing a recognizable structural motif, such as the helix-turn-helix or the zinc-finger. These motifs are compact structures that dock against the DNA double helix. Another DNA recognition motif, found in a highly conserved family of eukaryotic transcription factors including C/EPB, Fos, Jun and CREB, consists of a coiled-coil dimerization element the leucine-zipper and an adjoining basic region which mediates DNA binding. Here we describe circular dichroism and 1H-NMR spectroscopic studies of another family member, the yeast transcriptional activator GCN4. The 58-residue DNA-binding domain of GCN4, GCN4-p, exhibits a concentration-dependent alpha-helical transition, in accord with previous studies of the dimerization properties of an isolated leucine-zipper peptide. The GCN4-p dimer is approximately 70% helical at 25 degrees C, implying that the basic region adjacent to the leucine zipper is largely unstructured in the absence of DNA. Strikingly, addition of DNA containing a GCN4 binding site (AP-1 site) increases the alpha-helix content of GNC4-p to at least 95%. Thus, the basic region acquires substantial alpha-helical structure when it binds to DNA. A similar folding transition is observed on GCN4-p binding to the related ATF/CREB site, which contains an additional central base pair. The accommodation of DNA target sites of different lengths clearly requires some flexibility in the GCN4 binding domain, despite its high alpha-helix content. Our results indicate that the GCN4 basic region is significantly unfolded at 25 degrees C and that its folded, alpha-helical conformation is stabilized by binding to DNA.

Extracts (0.2 M NaCl) of HeLa cells support replication of DNA containing simian virus 40 (SV40) origin in the presence of SV40 large tumor (T) antigen. The reaction leads to the accumulation of high molecular weight products that represent DNA containing one parental strand and one progeny strand as well as duplex molecules that contain both strands derived from the input deoxynucleoside triphosphates. Mid-log-phase cultures (10 liters, 5 x i05 cells per ml) were harvested by centrifugation and the cell pellet was washed with 200 ml of ice-cold phosphate-buffered saline (137 mM NaCl/2.7 mM KC1/10.6 mM Na2HPO4/1.4 mM NaH2PO4).The cells were then washed once with 100 ml of cold hypotonic buffer (20 mM Hepes, pH 7.5/5 mM KCl/1.5 mM MgCl2/1 mM dithiothreitol) and resuspended in 30 ml of the same buffer. After swelling on ice for 10 min, the cells were disrupted by Dounce homogenization (20 strokes, B pestle). The lysate then was adjusted to 0.2 M NaCl and immediately centrifuged at 50,000 x g for 30 min. After dialysis for 3 hr against one change of buffer A (20 mM Hepes, pH 7.5/1 mM dithiothreitol/0.1 mM EDTA/10% (vol/vol) glycerol/S0 mM NaCl), the extract was clarified by centrifugation at 50,000 x g for 30 min and stored in aliquots at -80'C.DNA 5710The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Simian virus 40 (SV40) DNA replication dependent on the SV40 origin of replication and the SV40 large tumor (T) antigen has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, these components included the DNA polymerase G-primase complex, topoisomerase I, and a fraction that contained a single-stranded DNA binding protein. The latter protein, which sediments at 5.1 S on glycerol gradients and copurifles with two major protein species of 72 and 76 kDa, was isolated solely by its ability to support SV40 DNA replication. The purified system retained the species-speciflic DNA polymerase a-primase requirement previously observed with crude fractions; the complex from HeLa cells supported SV40 replication, whereas that from calf thymus and mouse cells did not. DNA containing the polyomavirus origin of replication was replicated in a system containing polyomavirus T antigen, the HeLa single-stranded DNA binding protein-containing fraction, and DNA polymerase primase complex from mouse, but not HeLa, cells. While crude fractions yielded closed circular duplex DNA, none was detected with the purified system. Nevertheless, the addition of a crude fraction to the purified system yielded closed circular monomer products.Replication of simian virus 40 (SV40) DNA requires only one virus-encoded protein, large tumor antigen (T antigen); initiates within a unique, well-defined origin sequence; proceeds bidirectionally; and terminates in a manner thought to be analogous to that utilized by the host chromosome (1, 2). Replication occurs on a template that is associated with nucleosomes in a structure resembling cellular chromatin (3). Thus, the study of SV40 and presumably cellular DNA replication should be facilitated by the recent development of in vitro systems that reproduce many key aspects of SV40 DNA replication in vivo (4)(5)(6)(7)(8). By using such systems, the DNA sequences required 'for origin function in vitro have been identified (9, 10), the roles of the complex of DNA polymerase a (pol a) and primase in viral replication and host species specificity have been investigated (11), and a system has been described whereby newly replicated DNA is assembled into a chromatin-like structure (12).Genetic and biochemical analyses of prokaryotic systems have revealed a number of activities directly involved in the enzymatic process of DNA replication: origin-specific binding activity, priming and deoxynucleotide polymerizing activities, helix unwinding activity, single-stranded DNA binding protein (SSB) to maintain the DNA in an unwound configuration, primer removal activity, DNA ligase, activities that relieve torsional strain accumulating ahead of the replication fork and resolve daughter molecules, and factors that modify either the template or one of the activities mentioned above to increase their efficiency (13). With these studies in mind, we have purified enzymes thought to be required for SV40 (and cellular) DNA replication in vivo and used the...

We have analyzed the DNA sequence requirements for TATA element function by assaying the transcriptional activities of 25 promoters, including those representing each of the 18 single-point mutants vitro (25, 32). One of these, TFIID, binds the TATA element, and this binding is required for its activity (10,27,28). Owing to the apparent instability of this activity during purification, it has not been possible to obtain preparations of mammalian TFIID of adequate purity for extensive biochemical characterization. However, the yeast Saccharomyces cerevisiae contains a 26-kilodalton (kDa) protein that can substitute for mammalian TFIID in DNA binding and in transcription reactions reconstituted with the other HeLa factors (4, 6). With this reconstitution assay, yeast TFIID has been purified to near homogeneity, and the gene encoding this protein has been cloned (5,11,14,22,34