Mark Ptashne

Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli

A strong promoter has been cloned on a series of plasmid vectors that facilitate the expression of cloned genes. This promoter, named tac [first described by DeBoer et al. (in Rodriguez, R.L. and Chamberlin, M.J. (Eds.),Promoters, Structure and Function. Praeger, New York, 1982, pp. pp. 462-481)] contains the -10 region of the lacUV5 promoter and the -35 region of the trp promoter. Our vectors contain various cloning sites followed by transcription termination signals. In addition, we describe plasmids that facilitate the conversion of the lac promoter to the stronger tac promoter. Thus, preexisting gene fusions using the lac or the lacUV5 promoter can be readily converted to tac promoter gene fusions without changing the ribosome-binding site (RBS). The tac promoter is repressed in lacIQ strains and can be induced by isopropylthio-beta-D-galactoside (IPTG). Studies of expression of the cI repressor of bacteriophage lambda show that the tac promoter is at least five times more efficient than the lacUV5 promoter. Under optimal conditions lambda repressor constitutes up to 30% of the total cellular protein.

Deletion analysis of GAL4 defines two transcriptional activating segments

We describe the activities of a wide array of deletion mutants of GAL4, a yeast transcriptional activator. We identify two short regions of GAL4, each of which activates transcription when fused to the DNA-binding region of the molecule. Very large portions of GAL4 are not required for gene activation.

A new class of yeast transcriptional activators

We describe yeast transcriptional activators encoded by E. coli genomic DNA fragments fused to the coding sequence of the DNA-binding portion of GAL4. All of the new activating sequences that we have analyzed, like those of GAL4 and GCN4, are acidic; most of these sequences show no obvious sequence homology when compared with the identified activating regions of GAL4 and GCN4 or among themselves. We also describe a fusion protein that contains no yeast protein sequence but activates transcription in yeast.

Specific DNA binding of GAL4, a positive regulatory protein of yeast

We show by the following series of experiments that the yeast positive regulatory protein GAL4 binds to four sites in the upstream activating sequence UASG to activate transcription of the adjacent GAL1 and GAL10 genes. GAL4 protein expressed in E. coli protected guanine residues in UASG from methylation by dimethyl sulfate. The same set of protections was seen in vivo in yeast and depended on the GAL4+ allele. This protection pattern is consistent with the idea that GAL4 protein binds to four related 17 bp sequences, each of which displays approximate 2-fold rotational symmetry. A single near-consensus synthetic 17 bp oligonucleotide, installed in front of the yeast GAL1 or CYC1 transcription units, conferred a high level of galactose inducibility upon these genes. Further experiments suggest that one mechanism of glucose repression is inhibition of the binding of GAL4 protein to DNA.

A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor

We describe a new protein that binds to DNA and activates gene transcription in yeast. This protein, LexA-GAL4, is a hybrid of LexA, an Escherichia coli repressor protein, and GAL4, a Saccharomyces cerevisiae transcriptional activator. The hybrid protein, synthesized in yeast, activates transcription of a gene if and only if a lexA operator is present near the transcription start site. Thus, the DNA binding function of GAL4 can be replaced with that of a prokaryotic repressor without loss of the transcriptional activation function. These results suggest that DNA-bound LexA-GAL4 and DNA-bound GAL4 activate transcription by contacting other proteins.

Negative effect of the transcriptional activator GAL4.

The yeast transcriptional activator GAL4 binds specific sites on DNA to activate transcription of adjacent genes1–5. The distinct activating regions of GAL4 are rich in acidic residues and it has been suggested that these regions interact with another protein component of the transcriptional machinery (such as the TATA-binding protein or RNA polymerase II) while the DNA-binding region serves to position the activating region near the gene6,7,8. Here we show that various GAL4 derivatives, when expressed at high levels in yeast, inhibit transcription of certain genes lacking GAL4 binding sites, that more efficient activators inhibit more strongly and that inhibition does not depend on the DNA-binding domain. We suggest that this inhibition, which we call squelching, reflects titration of a transcription factor by the activating region of GAL4.

Recognition of a DNA operator by the repressor of phage 434: a view at high resolution.

The repressors of temperate bacteriophages such as 434 and lambda control transcription by binding to a set of DNA operator sites. The different affinity of repressor for each of these sites ensures efficient regulation. High-resolution x-ray crystallography was used to study the DNA-binding domain of phage 434 repressor in complex with a synthetic DNA operator. The structure shows recognition of the operator by direct interactions with base pairs in the major groove, combined with the sequence-dependent ability of DNA to adopt the required conformation on binding repressor. In particular, a network of three-centered bifurcated hydrogen bonds among base pairs in the operator helps explain why 434 repressor prefers certain sites over others. These bonds, which stabilize the conformation of the bound DNA, can form only with certain sequences.

Cooperative binding of λ repressors to sites separated by integral turns of the DNA helix

Abstract λ repressors bind cooperatively to adjacent pairs of operator sites. Here we show that repressors bind cooperatively to pairs of operator sites whose centers have been separated by five or six turns of the helix. No cooperativity is observed when the centers of these sites are on opposite sides of the DNA helix. Cooperativity depends upon the same part of the protein (the carboxyl domain) that mediates cooperativity when the sites are adjacent. As the repressors bind, the DNA between the sites becomes alternately sensitive and resistant to DNAase I cleavage at half turn intervals. We suggest that when repressors bind cooperatively to separated sites, the DNA forms a loop, thus allowing the two repressors to touch.

Contact with a Component of the Polymerase II Holoenzyme Suffices for Gene Activation

In yeast strains bearing the point mutation called GAL11P (for potentiator), certain GAL4 derivatives lacking any classical activating region work as strong activators. The P mutation confers upon GAL11, a component of the RNA polymerase II holoenzyme, the ability to interact with a portion of the dimerization region of GAL4. The region of GAL11 affected by the P mutation is evidently functionally inert in ordinary cells, suggesting that this mutation is of no functional significance beyond creating an artificial target for the GAL4 dimerization fragment. From these observations and further analyses of GAL11, we propose that a single activator-holoenzyme contact can trigger gene activation simply by recruiting the latter to DNA.

The carboxy-terminal 30 amino acids of GAL4 are recognized by GAL80.

In wild-type yeast the action of the transcriptional activator GAL4 is inhibited by GAL80, and galactose relieves this inhibition. We show that deletion mutants of GAL4 lacking 30 amino acids of the carboxyl terminus activate transcription constitutively, whereas other deletion mutants bearing the carboxy-terminal 30 amino acids are inhibited by GAL80. Moreover, GAL4 fragments bearing these 30 amino acids, when expressed from a strong promoter on multicopy plasmids, free the endogenous GAL4 from inhibition by GAL80. These and other results suggest that GAL80 recognizes the carboxy-terminal 30 amino acids of GAL4, forming a complex that, though bound to DNA, does not activate transcription.