Biomedical subjects

M Ptashne

Publications and source records attributed to M Ptashne.

At least 73 records · Page 4Linked to original sources

PubMed1988

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.

Base Composition↗

PubMed1988

Converting a eukaryotic transcriptional inhibitor into an activator.

GAL80, an inhibitor of the yeast transcriptional activator GAL4, is converted into an activator by inserting an acidic activating sequence into it. This hybrid activator does not bind to DNA directly, but is brought to DNA by interacting with a derivative of GAL4 that interacts with both DNA and GAL80.

DNA, Fungal↗

PubMed1988

Structure of a phage 434 Cro/DNA complex.

Comparison of the crystal structure of a complex of the phage 434 Cro protein and a synthetic DNA operator with the complex of the same operator and the 434 repressor DNA-binding domain shows different DNA conformations in the two structures. Binding of the protein determines the precise conformation of the DNA in each case.

Bacteriophages↗

PubMed1988

How eukaryotic transcriptional activators work.

A specific protein, bound to DNA, can activate transcription of a wide array of genes in many eukaryotes. Further analysis suggests a general outline for how eukaryotic transcriptional activators function and are controlled.

Animals↗

PubMed1988

GAL4-VP16 is an unusually potent transcriptional activator.

Recent work has defined a class of transcriptional activators, members of which activate transcription in yeast, plant, insect and mammalian cells. These proteins contain two parts: one directs DNA binding and the other, called the activating region, presumably interacts with some component of the transcriptional machinery. Activating regions are typically acidic and require some poorly-understood aspect of structure, probably at least in part an alpha-helix. Here we describe a new member of this class, formed by fusing a DNA-binding fragment of the yeast activator GAL4 to a highly acidic portion of the herpes simplex virus protein VP16 (ref. 11; also called Vmw65). VP16 activates transcription of immediate early viral genes by using its amino-terminal sequences to attach to one or more host-encoded proteins that recognise DNA sequences in their promoters. We show that the hybrid protein (GAL4-VP16) activates transcription unusually efficiently in mammalian cells when bound close to, or at large distances from the gene. We suggest that the activating region of VP16 may be near-maximally potent and that it is not coincidental that such a strong activator is encoded by a virus.

Animals↗

PubMed1988

GAL4 derivatives function alone and synergistically with mammalian activators in vitro.

We show that derivatives of the yeast transcriptional activator GAL4, synthesized in and purified from E. coli, stimulate transcription of a mammalian gene (the adenovirus E4 gene) in a HeLa cell nuclear extract. Stimulation depended upon GAL4 binding sites inserted in the template. When the GAL4 sites were placed immediately upstream of the E4 TATA box, GAL4 stimulated efficiently. When the GAL4 sites were further upstream from TATA, however, efficient stimulation by GAL4 required, in addition, a site for a mammalian transcriptional activator immediately upstream of TATA. Under these conditions, the GAL4 derivatives functioned synergistically with the proximally bound activator. Previous experiments have defined two "activating regions" in GAL4, and our current experiments define a third, whose function is observed in vitro but not in vivo.

Adenoviruses, Human↗

PubMed1988

Negative effect of the transcriptional activator GAL4.

The yeast transcriptional activator GAL4 binds specific sites on DNA to activate transcription of adjacent genes. 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 gene. 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.

Binding Sites↗

PubMed1988

Yeast activators stimulate plant gene expression.

GAL4 is a transcriptional activator found in yeast. Two distinct functions of the protein are required for its activity: one directs sequence-specific DNA binding, and another interacts with some other component of the transcriptional machinery, for example, RNA polymerase II or a TATA-binding protein. Two short regions of GAL4 function as 'activating sequences' when attached to the DNA-binding portion of GAL4 and these regions can be replaced by a large number of peptides encoded by Escherichia coli genomic DNA fragments or by a synthetic peptide designed to form an amphiphilic alpha-helix. All of these activating sequences, like that found in another yeast activator, GCN4 bear an excess negative charge. GAL4 and its derivatives that are active in yeast stimulate transcription in mammalian cells when GAL4 binding sites are introduced upstream of a mammalian gene; similarly, GAL4 activates transcription in Drosophila cells. Here we show that GAL4 derivatives stimulate gene expression in plant cells.

Acetyltransferases↗

PubMed1988

Turning lambda Cro into a transcriptional activator.

According to our present understanding, lambda repressor bound to DNA stimulates transcription by touching RNA polymerase bound at an adjacent promoter. The part of repressor required for activation was identified in part by the isolation of mutants specifically impaired in transcriptional activation. The amino acids of repressor altered in these "positive control" mutants lie in an acidic patch on the surface of repressor that is closely apposed to RNA polymerase. In this study, we show that this "activating patch" of repressor is sufficient for transcriptional activation in another sequence context. We transfer this activating patch onto the surface of lambda Cro, a protein normally unable to activate transcription, and show that the modified Cro is a transcriptional activator. In addition, we provide evidence that the repressor protein of phage 434 also activates transcription using an activating patch similar to that of lambda repressor.

Bacteriophage lambda↗

PubMed1988

GAL4 activates transcription in Drosophila.

GAL4 is a yeast regulatory protein that binds to specific sites within a DNA sequence called UASG (galactose upstream activating sequence) and activates transcription of linked genes. This activation requires two functions of the protein: a DNA binding domain located near the amino terminus, and one or more 'activating regions'. The 'activating regions' are highly acidic (see also ref. 12) and can be replaced, for example, by a short peptide designed to form a negatively charged, amphipathic alpha-helix. GAL4, as well as deletion derivatives bearing one or more 'activating regions' attached to the DNA binding domain, activates transcription in cultured mammalian cells from mammalian promoters linked to a UASG (refs 14, 15). Here we show that GAL4, when expressed in particular tissues of Drosophila larvae, stimulates tissue-specific transcription of a Drosophila promoter linked to GAL4 binding sites.

Animals↗

PubMed1988

GAL4 activates gene expression in mammalian cells.

GAL4, a protein that activates transcription in yeast, is shown to activate the mouse mammary tumor virus promoter in mammalian cells. Activation depends upon a GAL4-binding sequence inserted upstream of the gene. Deletion mutants of GAL4 bearing one or both of the "activating regions" required for activation in yeast also activate transcription in mammalian cells. A derivative of GAL4 that binds to DNA but cannot activate transcription in yeast also fails to activate transcription in mammalian cells. We also show that GAL4 and the glucocorticoid receptor activate the mouse mammary tumor virus promoter synergistically.

Acetyltransferases↗

PubMed1988

Recognition of DNA sequences by the repressor of bacteriophage 434.

The structure of a complex between the DNA-binding domain of phage 434 repressor and a 14 base-pair synthetic DNA operator reveals the molecular interactions important for sequence-specific recognition. A set of contacts with DNA backbone, notably involving hydrogen bonds between peptide-NH groups and DNA phosphates, position the repressor and fix the DNA configuration. Direct interactions between amino acid side chains and DNA bases involve nonpolar van der Waals contacts as well as hydrogen bonds. The structures of the repressor domain and of the 434 cro protein are extremely similar. There appear to be no major conformational changes in the proteins when they bind to DNA.

Bacteriophages↗

PubMed1988

No strict alignment is required between a transcriptional activator binding site and the "TATA box" of a yeast gene.

GAL4 is a transcriptional activator of the galactose metabolism genes in the yeast Saccharomyces cerevisiae. We show that GAL4 expressed in yeast activated transcription equally well when a single GAL4 binding site was placed at any of nine positions upstream of the GAL1 (galactokinase gene) "TATA box." We chose a sufficient number of positions for the binding site to ensure that, in several of these positions, GAL4 was on the opposite side of the DNA helix with respect to the TATA box. Smaller GAL4 derivatives were similar to wild-type GAL4 in that they also activated transcription in a manner independent of the side of the DNA helix they bound with respect to the TATA box. Unlike wild-type GAL4, however, these smaller GAL4 derivatives activated transcription better when we placed a binding site progressively closer to the TATA box over a distance of 34 base pairs.

Bacterial Proteins↗

PubMed1988

DNA twisting and the affinity of bacteriophage 434 operator for bacteriophage 434 repressor.

The affinity of the Escherichia coli phage 434 operator for phage 434 repressor is affected by changes in the sequence of the noncontacted base pairs near the operator's center. The results presented here show that base composition near the center of the operator affects the operator's affinity for repressor by altering the ease with which the operator can be overtwisted into the proper configuration for complex formation. We show that both DNA flexibility and repressor flexibility influence the strength of the repressor-operator interaction: an operator with a single-strand nick at its center has a higher affinity for repressor than does the intact operator: and a repressor bearing a mutation that results in a relaxed dimer interaction is less sensitive than is wild type to changes in the flexibility of the operator. We show that the effect of noncontacted base pairs on operator affinity is independent of the slight overall bend of the operator seen in the repressor-operator complex. Central sequence effects on affinity for repressor are independent of the identity of adjacent base pairs, suggesting that the structure of the individual base pairs, not interactions between them, are responsible for the different torsional rigidities of different operators.

Base Composition↗

PubMed1988

A repressor heterodimer binds to a chimeric operator.

Replacement of the solvent-exposed residues of the DNA recognition helix of the 434 repressor with the corresponding residues of the P22 repressor generates a hybrid protein, 434R[alpha 3(P22R)], which binds specifically to P22 operators. We show here that a new DNA-binding specificity is generated by combining 434 and 434R[alpha 3(P22R)] repressor monomers to form a heterodimer. The heterodimer specifically recognizes a chimeric P22/434 operator that lacks two-fold rotational symmetry.

Base Sequence↗

PubMed1988

Cooperative DNA binding of the yeast transcriptional activator GAL4.

The yeast transcriptional regulatory protein GAL4 binds to four sites in the GAL upstream activating sequence and stimulates transcription of the adjacent GAL1 and GAL10 genes. We show here that binding to at least two of these sites is cooperative in vivo. We also measure stimulation of transcription by pairs of GAL4 binding sites and find that the activities of low-affinity binding sites combine synergistically, whereas the activities of high-affinity binding sites combine only additively. We suggest that the synergistic transcriptional stimulatory activity of the naturally occurring GAL4 binding sites is solely a manifestation of cooperative binding of GAL4 protein to DNA and that the activity of a GAL upstream activating sequence is roughly proportional to the number of GAL4 molecules bound.

Binding Sites↗

PubMed1987

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.

Amino Acid Sequence↗

PubMed1987

Mutants of GAL4 protein altered in an activation function.

Activating region I of GAL4 has been defined as a region 48 amino acids long which, when attached to GAL4's DNA-binding domain, activates transcription in yeast. Here we describe mutants bearing changes in and around this highly acidic activating region. We find mutations that increase the activation function invariably increase the acidity of the region and some but not all of the mutations that decrease the activation function decrease the acidity of the region.

Amino Acid Sequence↗