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Mark Ptashne

Publications and source records attributed to Mark Ptashne.

16 recordsLinked to original sources
PubMed2005

Transcriptional activating regions target attached substrates to a cyclin-dependent kinase.

The yeast cyclin-dependent kinase Srb10 phosphorylates various transcriptional activators as they activate transcription, and acidic transcriptional activating domains found on several activators directly bind Srb10. Here we show that the interaction between Srb10 (with its associated cyclin Srb11) and each of several different activating regions, in vitro, leads to the phosphorylation of peptide sequences attached to but outside of the activating regions themselves. In some cases, residues within the activating regions are also phosphorylated. The results define a mechanism by which a kinase is recruited to alternate substrates with diverse physiological consequences.

Amino Acid Sequence↗
PubMed2004

Activation of the Gal1 gene of yeast by pairs of 'non-classical' activators.

Eukaryotic transcriptional activators work by recruiting to DNA the transcriptional machinery, including protein complexes required for chromatin modification, transcription initiation, and elongation. Which of these complexes must be directly recruited to trigger transcription? We test various "non-classical" transcription activators (comprising a component of the transcriptional machinery fused to a DNA binding domain) for their abilities to activate transcription of a chromosomally integrated reporter in yeast. Among these newly constructed fusion proteins, none efficiently activated transcription when working on its own. However, in several instances transcription was activated by a pair of such fusion proteins tethered to adjacent sites on DNA. In each of these cases, one fusion protein bore a component of the SAGA complex, and the other bore a component of the Mediator complex. Transcription was also activated by certain tripartite fusion proteins comprising a Mediator and a SAGA component fused to a DNA binding domain. The results are consistent with the finding that the classical activator Gal4, working at the GAL1 promoter, activates transcription by (at least in part) independently recruiting SAGA and Mediator.

DNA-Binding Proteins↗
PubMed2003

Regulated recruitment and cooperativity in the design of biological regulatory systems.

What distinguishes a man from a mouse is not so much different proteins, but rather the appearance of common proteins (etc.) at different times and positions in the developing organisms. Thus specific genes are transcribed or repressed, proteins degraded or stabilized, RNA transcripts spliced one way or another, and so on. These are examples of 'regulatory' decisions. A rather simple mechanism - called regulated recruitment - lies at the heart of many of these regulatory decisions.

Enzyme Activators↗
PubMed2003

RNA sequences that work as transcriptional activating regions.

We describe a set of RNA molecules that work as transcriptional activators when tethered to DNA. These RNA activating regions were found amongst a randomized set of molecules bearing variants of a 10 nt loop attached to an RNA stem. The various RNA activating regions all bear an identical five- residue sequence with an interspersed sixth residue. The result shows that although all natural activating regions characterized thus far are peptidic, this function can be served by other kinds of moieties as well.

Base Sequence↗
PubMed2003

Independent recruitment in vivo by Gal4 of two complexes required for transcription.

We use a modified form of ChIP to analyze the recruitment of seven sets of proteins to the yeast GAL genes upon induction. We resolve three stages of recruitment: first SAGA, then Mediator, and finally Pol II along with four other proteins (including TBP) bind the promoter. In a strain lacking SAGA, Mediator is recruited with a time course indistinguishable from that observed in wild-type cells. Our results are consistent with the notion that a single species of activator, Gal4, separately contacts, and thereby directly recruits, SAGA and Mediator.

Binding Sites↗
PubMed2002

Design of artificial transcriptional activators with rigid poly-L-proline linkers.

Typical eukaryotic transcriptional activators are composed of distinct functional domains, including a DNA binding domain and an activating domain. Artificial transcription factors have been designed wherein the DNA binding domain is a minor groove DNA binding hairpin polyamide linked by a flexible tether to short activating peptides, typically 16-20 residues in size. In this study, the linker between the polyamide and the peptide was altered in an incremental fashion using rigid oligoproline "molecular rulers" in the 18-45 A length range. We find that there is an optimal linker length which separates the DNA and the activation region for transcription activation.

Amino Acid Sequence↗
PubMed2002

Transcriptional activating regions target a cyclin-dependent kinase.

Several yeast activators are phosphorylated by SRB10, a cyclin-dependent kinase associated with the transcriptional machinery. Sites of phosphorylation are found outside the activating region in each case, and the modification has different physiological consequences in different cases. We show here that certain acidic transcriptional activating regions contact SRB10 as assayed both in vivo and in vitro. The interaction evidently positions each activator, as it activates transcription, so that it gets phosphorylated by SRB10, and thus a common mechanism targets disparate substrates to the kinase.

Cyclin-Dependent Kinase 8↗
PubMed2002

Responses of four yeast genes to changes in the transcriptional machinery are determined by their promoters.

Many yeast genes are distinguished by their specific requirements for different components of the transcriptional machinery. Here we examine four genes that fall into two classes as defined by their dependence on specific components of the transcriptional machinery. We describe a series of hybrid constructs, each of which bears activator binding sites that are associated with a promoter other than that with which they are usually affiliated. We examine expression of these reporters in strains bearing three modifications of the transcriptional machinery. Our results indicate that, in each of these cases, the promoter (and not the activator) determines which components of the transcriptional machinery are required. These and additional results, including those of others, clarify how disparate activators can work at many different promoters.

Genes, Fungal↗
PubMed2002

A target essential for the activity of a nonacidic yeast transcriptional activator.

P201 is a short (eight-residue) nonacidic peptide that comprises a strong transcriptional activating region when tethered to DNA in yeast. Here we identify the mediator protein Gal11 as an essential target of P201. Deletion of Gal11, which modestly decreases activation elicited by the typical acidic yeast activator, abolishes activation by DNA-tethered P201. A point mutation in Gal11, which has no effect on other Gal11 functions, also greatly diminishes activation by DNA-tethered P201. P201 binds to a fragment of Gal11 in vivo and in vitro, and the interaction is diminished by mutations in either component that decrease activation in vivo. P201, unlike the typical yeast acidic activating region, does not work in mammalian cells, which is consistent with the notion that the unique target of P201 (Gal11) is absent from mammalian cells.

Fungal Proteins↗
PubMed2002

Telomere looping permits repression "at a distance" in yeast.

In yeast, unlike in higher eukaryotes, transcriptional activators and repressors do not normally work when bound to DNA at large distances (over 500 base pairs) from the gene and, in particular, when positioned downstream of the gene. This restriction is relieved for a transcriptional activator if a gene bearing an activator binding site is placed near a yeast telomere. The explanation proposed is that the folded structure found at the telomere helps appose the DNA-bound activator with proteins binding to the promoter so that recruitment of the transcriptional machinery can be effected "at a distance". Here, we show that a repressor, Tup1, works when tethered to DNA downstream of, and some 1.5-kb from, the gene when the construct is placed near a yeast telomere. The effect, observed with activated as well as basal transcription, is eliminated by deletion of Sir3. These and other results indicate that DNA-tethered Tup1 represses by interacting with some component of the transcriptional machinery binding to the promoter, an interaction that is facilitated by the preformed loop at the telomere.

Base Sequence↗
PubMed2002

The TBP-inhibitory domain of TAF145 limits the effects of nonclassical transcriptional activators.

Many genes in bacteria and eukaryotes are activated by "regulated recruitment". According to that picture, a transcriptional activator binds cooperatively to DNA with the transcriptional machinery, and the constitutively active polymerase then spontaneously transcribes the gene. An important class of experiments that helped develop this model is called the "activator by-pass" experiment. In one version of such an experiment, the ordinary activator-transcriptional machinery interaction is replaced by a heterologous interaction. For example, fusing any of several DNA binding domains to Gal11, a component of the yeast mediator complex, creates a powerful activator of genes bearing the corresponding DNA binding sites. Here, we describe a simple modification of the yeast transcriptional machinery that extends the success of similar experiments involving other mediator components. The results reinforce parallels between regulation of enzymes involved in transcription and in other cellular processes.

Base Sequence↗