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M Ptashne

Publications and source records attributed to M Ptashne.

At least 19 recordsLinked to original sources
PubMed1992

Multiple mechanisms mediate glucose repression of the yeast GAL1 gene.

Several mechanisms contribute to the glucose repression of the GAL1 gene in Saccharomyces cerevisiae. We show that one mechanism involves the transcriptional down-regulation of the GAL4 gene and a second requires the GAL80 gene. We also examine the contribution of cis-acting negative elements in the GAL1 promoter to glucose repression. In an otherwise wild-type strain disruption of any one of these three mechanisms alleviates repression of GAL1 only 2- to 4-fold. However, in the absence of the other two mechanisms the transcriptional down-regulation of GAL4 is sufficient to repress GAL1 expression 40- to 60-fold and the GAL80-dependent mechanism is sufficient to repress GAL1 expression 20- to 30-fold. These first two mechanisms constitute a functionally redundant system of repression and both must be disrupted in order to abolish glucose repression of GAL1. In contrast, negative elements in the GAL1 promoter are effective in repressing GAL1 expression 2- to 4-fold in glucose medium only when at least one of the other two mechanisms of repression is present. Thus, glucose repression of GAL1 is mediated primarily by the first two mechanisms, whereas the third mechanism supplements repression severalfold.

DNA Mutational Analysis↗
PubMed1992

DNA recognition by GAL4: structure of a protein-DNA complex.

A specific DNA complex of the 65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4, has been analysed at 2.7 A resolution by X-ray crystallography. The protein binds as a dimer to a symmetrical 17-base-pair sequence. A small, Zn(2+)-containing domain recognizes a conserved CCG triplet at each end of the site through direct contacts with the major groove. A short coiled-coil dimerization element imposes 2-fold symmetry. A segment of extended polypeptide chain links the metal-binding module to the dimerization element and specifies the length of the site. The relatively open structure of the complex would allow another protein to bind coordinately with GAL4.

Amino Acid Sequence↗
PubMed1991

GAL4 is phosphorylated as a consequence of transcriptional activation.

GAL4 protein isolated from yeast in which it is active is phosphorylated predominantly on two different serine residues. One of these was identified as Ser-837; substitution of this residue for alanine has no detectable effect on transcriptional activation by GAL4. Phosphorylation at Ser-837 requires that both the DNA binding and transcriptional activation functions be intact. We propose that some phosphorylations of GAL4, including that at Ser-837, occur concomitantly with activation of transcription.

Amino Acid Sequence↗
PubMed1991

Generating yeast transcriptional activators containing no yeast protein sequences.

We previously reported that roughly 1% of the short peptides encoded by Escherichia coli genomic DNA fragments act as transcriptional activating regions in yeast when fused to GAL4(1-147), a DNA-binding portion of the yeast transcriptional activator GAL4 (ref. 1). Struhl questioned the conclusion that we had identified new transcriptional activating sequences that function in the absence of yeast transcriptional activating sequences. His criticism was based on two considerations: first, GAL4(1-147) contains an acidic segment (and subsequent experiments have shown that this region contains a weak activating region in vitro); second, attempts to isolate new activating regions failed when the DNA-binding domain of a bacterial repressor, LexA(1-87), was used as the DNA-binding unit. We report here a repeat of our original experiment using the complete LexA molecule LexA(1-202) as the DNA-binding region, instead of GAL4(1-147) or LexA(1-87). We find that, as in the original experiment, about 1% of the short peptides encoded by E. coli genomic fragments act as transcriptional activating regions when fused to intact LexA. All of the new activating regions whose sequences we determined bore an excess of acidic amino acids (see Table 1).

Amino Acid Sequence↗
PubMed1990

GAL11P: a yeast mutation that potentiates the effect of weak GAL4-derived activators.

A mutant yeast in which a weak GAL4-derived activator functions as a strong activator bears a single mis-sense mutation in GAL11 (a.k.a. SPT13). The first 74 amino acids of GAL4, including the zinc-dependent DNA binding region, attached to an acidic activating sequence, are sufficient to respond both to GAL11 and to our mutant GAL11P (potentiator). PPR1, a yeast activator with a similar zinc finger sequence, also responds to GAL11 and to GAL11P, whereas regulators bearing unrelated DNA binding motifs do not. GAL11 itself works as a strong activator when tethered to DNA by fusion to the bacterial LexA protein, and deletion of GAL11 is known to cause a 5- to 10-fold reduction in GAL4 activity. We suggest that a complex of GAL4 and GAL11 constitutes a particularly strong activator; evidence that the putative GAL4-GAL11 complex ordinarily forms preferentially on DNA suggests a biological rationale for GAL11 action.

Amino Acid Sequence↗
PubMed1990

Activators and targets.

Proteins that activate genes are quite disparate in character; in particular, some work 'universally' and others do not. A simple model can accommodate most of the recently published results.

Adenovirus Early Proteins↗
PubMed1990

How different eukaryotic transcriptional activators can cooperate promiscuously.

A striking characteristic of many different eukaryotic transcriptional activators is their ability to activate gene expression synergistically. Thus, for example, the rat glucocorticoid receptor and the yeast activator GAL4 cooperatively activate transcription of a mammalian gene bearing binding sites for each of the proteins: activation by both activators is greater than the sum of the effects of each working alone. It would seem unlikely that these two proteins from such different organisms directly interact; rather, the idea has been suggested that these and at least some other eukaryotic activators can work synergistically by simultaneously touching some part of the transcriptional machinery. An important prediction of this idea is that synergy between two such activators would be seen under conditions where each is present at concentrations sufficiently high to saturate its site on DNA. In this paper we use transcription in vitro to confirm that prediction using a derivative of the yeast activator GAL4 and the mammalian transcription factor ATF. The accompanying paper describes a similar conclusion comparing the effects of singly and multiply bound GAL4 molecules.

Activating Transcription Factors↗
PubMed1990

A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives.

In prokaryotes and eukaryotes many gene activators work synergistically. For example, two dimers of lambda repressor interact to promote binding of these proteins to DNA, a reaction that is crucial at the repressor concentrations found in lysogens. In this case one of the bound dimers activates transcription, evidently by touching RNA polymerase. In another example, the yeast transcriptional activator GAL4, which can stimulate transcription in many eukaryotes, binds to multiple sites on DNA to activate transcription synergistically; the presence of two such sites can elicit a level of transcription more than twice that found with a single site. In this paper we show that synergistic activation by each of several GAL4 derivatives involves a mechanism different from that illustrated by the lambda repressor: multiple activator molecules can work synergistically under conditions in which their binding sites on DNA are saturated. The accompanying paper shows that under similar conditions of activator excess, GAL4 derivatives work synergistically with a heterologous mammalian gene activator. These results support the idea that eukaryotic activators can cooperate not by directly interacting but by simultaneously touching some component(s) of the transcriptional machinery.

Binding Sites↗
PubMed1990

A potent GAL4 derivative activates transcription at a distance in vitro.

Transcription of a typical eukaryotic gene by RNA polymerase II is activated by proteins bound to sites found near the beginning of the gene as well as to sites, called enhancers, located a great distance from the gene. According to one view, the primary difference between an activator that can work at a large distance and one that cannot is that the former bears a particularly strong activating region; the stronger the activating region, the more readily the activator interacts with its target bound near the transcriptional start site, with the intervening DNA looping out to accommodate the reaction. One alternative view is that the effect of proteins bound to enhancers might require some special aspect of cellular or chromosome structure. Consistent with the first view, an activator bearing an unusually potent activating region can stimulate transcription of a mammalian gene in a HeLa nuclear extract when bound as far as 1.3 kilobase pairs upstream or 320 base pairs downstream of the transcriptional start site.

Activating Transcription Factors↗
PubMed1990

Mutations that increase the activity of a transcriptional activator in yeast and mammalian cells.

Activating region I of GAL4 protein is a stretch of amino acids, positioned adjacent to the DNA-binding region, that activates transcription in yeast and, as we show here, in mammalian cells. Here we describe mutations located throughout a 65-amino acid region that increase the activation function of region I. Most of these mutations replace positively charged amino acids in the region with neutral ones, although we also describe substitutions at one position that do not alter the charge of the region. Mutations of region I that alter the activation function in yeast have similar effects on activation when assayed in mammalian cells. When individual mutations that raise the acidity of the activating region are recombined, the activities of the mutant proteins increase with increasing negative charge in both yeast and mammalian cells. These results extend and modify the correlation between acidity and activation and suggest that the requirements for a strong activating region are conserved in yeast and mammals.

Amino Acids↗
PubMed1989

P22 repressor mutants deficient in co-operative binding and DNA loop formation.

We show here, both in vivo and in vitro, that P22 repressor binds co-operatively to operator sites separated by an integral number of turns of the DNA helix. We measure this co-operativity in vivo using an assay in which repression of a promoter requires co-operative binding of P22 repressors to two separated (non-adjacent) operator sites. We report the isolation of mutant repressors that have high affinity for single operator sites, but are defective in co-operative binding. Six different mutants, all bearing single amino acid changes in the carboxyl domain, have been isolated. We purified the two mutants most deficient in co-operative binding, and found that they bind non-co-operatively in vitro to adjacent as well as to non-adjacent pairs of operator sites.

Bacteriophage lambda↗
PubMed1989

Rescue of bicoid mutant Drosophila embryos by bicoid fusion proteins containing heterologous activating sequences.

The maternal gene bicoid (bcd) determines pattern in the anterior half of the Drosophila embryo. It is reported here that the injection of bcd mutant embryos with messenger RNAs that encode proteins consisting of heterologous acidic transcriptional activating sequences fused to the DNA-binding portion of the bcd gene product, can completely restore the anterior pattern of the embryo.

Animals↗
PubMed1989

An amino-terminal fragment of GAL4 binds DNA as a dimer.

GAL4 is a yeast transcriptional activator protein that binds to specific 2-fold rotationally symmetric sites on DNA and stimulates transcription of the genes required for galactose catabolism. The DNA binding region of the protein is located within the first 74 amino acids and contains a "zinc finger" sequence motif. We show that a polypeptide comprising the first 147 amino acids of GAL4, designated GAL4 (1-147), binds DNA as a dimer in vitro. Although a protein containing only the first 74 amino acids, designated GAL4 (1-74), binds DNA specifically, its affinity is reduced relative to GAL4 (1-147). Addition of the strong dimerization domain of lambda repressor to GAL4 (1-74) generates a protein that binds as tightly as GAL4 (1-147). GAL4 (1-147) makes rotationally symmetric contacts with its recognition site when assayed by DNase I, exonuclease III and hydroxyl radical footprinting and by phosphate ethylation interference. Binding of GAL4 (1-147) in vitro requires either zinc or cadmium.

Amino Acids↗
PubMed1989

A single glutamic acid residue plays a key role in the transcriptional activation function of lambda repressor.

Previous experiments have suggested that negative charge is an important aspect of the activating region of lambda repressor as it is for at least one class of eukaryotic transcriptional activators. Here we randomize amino acids in the activating region of repressor and assay the function of over 100 variants. We find that acidic residues at the four solvent-exposed positions on the surface of an alpha helix (helix 2 in the structure) together comprise a strong activating region. Only one of these acidic residues, however, is critical for activation, and at this position glutamate is strongly preferred to aspartate. At the three remaining positions, certain uncharged residues (different ones at each position) function as well as or better than the acidic residues. Basic residues, however, are highly detrimental to function at all four positions. Our mutagenesis studies also suggest limitations on amino acid substitutions that allow formation of the helix-turn-helix DNA binding motif found in repressor and in many other DNA binding regulatory proteins.

Amino Acid Sequence↗
PubMed1989

Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

Base Sequence↗