We have constructed a system which allows systematic testing of repressor–operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette‐like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor.

The Drosophila HMG1-like protein DSP1 was identified by its ability to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion in yeast. We show here that DSP1 as well as its mammalian homolog hHMG2 bind to the mammalian protein SP100B and that SP100B in turn binds to human homologs of HP1. The latter is a Drosophila protein that is involved in transcriptional silencing. Each of these proteins represses transcription when tethered to DNA in mammalian cells. These results suggest how heterochromatin proteins might be recruited to specific sites on DNA with resultant specific effects on gene expression.The Drosophila protein Dorsal can act as a transcriptional activator or repressor depending on the promoter context. For example, in Drosophila, Dorsal activates the twist promoter but represses the zen promoter. A Dorsal-binding element taken from the zen promoter, called the ventral repression element, and placed upstream of an activated gene in a Drosophila embryo, mediates Dorsal-dependent repression of that gene (1). In Saccharomyces cerevisiae, however, Dorsal activates transcription from both the twist and zen promoters. DSP1, a member of the high mobility group1͞2 (HMG) family of non-histone chromosomal DNA-binding proteins, was isolated as a putative corepressor that inhibits Dorsal from activating the zen promoter but has no effect on Dorsal activation of a reporter bearing certain isolated Dorsal-binding sites (2). DSP1 interacts with Dorsal and with p50͞p65 heterodimer NF-B and binds cooperatively with these proteins to DNA (ref. 2 and J. Brickman and M.P., unpublished data).

One protein can activate some genes and repress others in the same cell. The Drosophila protein Dorsal (which, like the human protein NF-kappa B3, is a member of the Rel family of transcriptional activators) activates the twist gene and represses the zen gene in the ventral region of early embryos. Here we describe a Drosophila HMG1 protein, called DSP1 (dorsal switch protein), that converts Dorsal and NF-kappa B from transcriptional activators to repressors. This effect requires a sequence termed a negative regulatory element (NRE), found adjacent to Dorsal-binding sites in the zen promoter and adjacent to the NF-kappa B-binding site in the human interferon-beta (IFN-beta) enhancer. Previous studies have shown that another type of HMG protein, HMG I(Y), can stimulate NF-kappa B activity. Thus, the HMG-like proteins DSP1 and HMG I(Y) can determine whether a specific regulator functions as an activator or a repressor of transcription.

Proteins which recognize specific sequences of DNA play a fundamental role in the regulation of protein synthesis in all organisms. A particular helix of the bacterial protein lac repressor recognizes the bases in the major groove of the lac operator. We show that the first two residues of this recognition helix interact independently with two base pairs. This allows us in many cases to predict repression as an indicator of strength of the repressor-operator complex. Rules of recognition can be derived for 16 symmetric operators. They also apply to the gal repressor and possibly to other bacterial repressors.

The split-ubiquitin assay detects protein interactions in vivo. To identify proteins interacting with Gal4p and Tup1p, two transcriptional regulators, we converted the split-ubiquitin assay into a generally applicable screen for binding partners of specific proteins in vivo. A library of genomic Saccharomyces cerevisiae DNA fragments fused to the N-terminal half of ubiquitin was constructed and transformed into yeast strains carrying either Gal4p or Tup1p as a bait. Both proteins were C-terminally extended by the C-terminal half of ubiquitin followed by a modified Ura3p with an arginine in position 1, a destabilizing residue in the N-end rule pathway. The bait fusion protein alone is stable and enzymatically active. However, upon interaction with its prey, a native-like ubiquitin is reconstituted. RUra3p is then cleaved off by the ubiquitin-specific proteases and rapidly degraded by the N-end rule pathway. In both screens, Nhp6B was identified as a protein in close proximity to Gal4p as well as to Tup1p. Direct interaction between either protein and Nhp6B was confirmed by coprecipitation assays. Genetic analysis revealed that Nhp6B, a member of the HMG1 family of DNA-binding proteins, can influence transcriptional activation as well as repression at a specific locus in the chromosome of the yeast S. cerevisiae. The split-ubiquitin method is based on the ability of N ub and C ub , the N-and C-terminal halves of ubiquitin, to form a native-like ubiquitin (1). Ubiquitin-specific proteases (UBPs), present in the cytosol and nucleus of all eukaryotic cells, recognize the reconstituted ubiquitin, but not its halves, and cleave off a reporter protein, which had been linked to the C terminus of C ub . The split-ubiquitin assay (split-Ub) is designed to yield efficient association of N ub and C ub only if the two ubiquitin halves are linked to proteins that interact in vivo. The assay has been shown to detect interactions between cytosolic proteins, membrane proteins, and transient interactions that occur between transporter and substrate during protein translocation across the membrane of the endoplasmic reticulum in vivo (1-4). In addition, split-Ub can also be used to demonstrate interactions between transcription factors (5, 6) because, contrary to the two-hybrid system (7), it is not based on a transcriptional readout.The Saccharomyces cerevisiae GAL1 promoter is a wellstudied example of transcriptional regulation by nutrients. When the cells are grown in medium containing galactose as the sole carbon source, GAL1 is activated by Gal4p, which binds specifically to the GAL1 promoter. Gal4p interacts with the holoenzyme component Srb4p, thereby recruiting the transcription apparatus to the GAL1 promoter (8). If the carbon source is switched to glucose, the promoter is repressed by two independently operating mechanisms. Gal80p masks the activation domain of DNA-bound Gal4p, thereby preventing the recruitment of the transcription machinery (9). In addition, the cytosolic repressor Mig1p enters the nucleus (10). Mig1p b...