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In this article the author examines the scientific problem of gene regulation. He describes the various stages of his understanding of gene regulation, as well as insights from his colleagues who are also involved in the regulation of prokaryotic and eukaryotic genes. He also discusses the regulatory molecules called repressors that would turn off expression of specific genes.

In this article, the author discusses bacterial virus (phage) lambda's genetic switch which responds to environmental signals sent from other cells. It states that the switch also controls transition between patterns in response to extracellular signals. It says that the switch works by protein-protein and protein-DNA binding reaction.

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 cerevislae transcriptional activator. The hybrid protein, synthesized in yeast, activates transcription of a gene if and only if a IexA 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 contracting other proteins, [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

The author discusses the inaccuracy to label biological response modifiers in chromatin as epigenetics due to their responsive properties in being genetically recruited by specific transcription regulators. He states that gene regulatory changes can be derived from epigenetic development. The author notes that convincing evidence is still lacking for the self-perpetuating tendencies of biological modifiers.

Studies two mitogen-activated protein kinase signaling cascades in yeast to understand the specificity of kinases. Mechanism of action of the cascade involved in yeast mating; Osmolarity cascade activated in response to high salt concentrations in the medium; Elaboration of osmolarity response to a mating signal in cells bearing hybrid scaffold.

Examines the mechanism for the C6 zinc cluster family of yeast transcriptional activators. Ammonium-terminal sequences of GAL4, PUT3 and PPRI; Hybrids; Differences in DNA binding specificity; Diagrammatic representation; Observation of GAL4 bonding to CGG triplets.

The author reflects on epigenetics. He notes a definition of epigenetics as short-term heritable alterations in gene expression that are not due to mutations, presented in a book. He cites that states of gene expression are maintained as cells divice. He explains the association of active genes with acetylated nucleosomes.

This article presents information related to DNA binding. By 1985 we had clear ideas for how lambda regulatory proteins bind DNA and turn transcription on and off. Perhaps a new mode of binding to DNA was required, for example; perhaps activation at a distance requires special functions not represented among the lambda regulators; and so on. The lambda repressor binds without dramatically changing the structure of DNA. The protein is both an activator and a repressor of transcription, and so neither of these activities requires DNA conformational changes.

The article presents an obituary for Horace Judson, and author and a historian of molecular biology, is presented.

A letter to the editor is presented in response to the editorial on the scientific basis of epigenome project in the 2010 issue.

Summary: Simple binding interactions lie at the heart of disparate biological functions. Multiple negative and positive ‘add-ons’, often with small individual effects, make elementary systems that work, work better. Cancer illustrates various of these fundamental processes gone awry. [Copyright &y& Elsevier]

Summary: Yeast growing for a considerable time in glucose ‘remember’ a previous exposure to galactose, the inducer of its galactose-utilization (GAL) genes. This memory is conveyed by a cytoplasmically transmitted galactokinase working as a signal transducer. [Copyright &y& Elsevier]

Over the past few years we have seen an odd change, or extension, in the use of the word ‘epigenetic’ when describing matters of gene regulation in eukaryotes. Although it may generally be that it is not worth arguing over definitions, this is true only insofar as the participants in the discussion know what each other means. I believe the altered use of the term carries baggage from the standard definition that can have misleading implications. Here I wish to probe our use of language in this way, and to show how such a discussion leads to some more general considerations concerning gene regulation. [Copyright &y& Elsevier]

A recent experiment has replaced Cro, a crucial component of lambda''s genetic switch, with the lac repressor (plus two lac operators). The resulting hybrid phage is viable, but a subtle phenotypic defect explains a puzzle concerning the workings of the switch. [Copyright &y& Elsevier]

Presents an obituary for Ira Herskowitz, a scientist who died on April 28, 2003 of pancreatic cancer.

Induction of transcription of the GAL genes of yeast by galactose is a multistep process: Galactose frees the activator Gal4 of its inhibitor, Gal80, allowing Gal4 to recruit proteins required to transcribe the GAL genes. Here, we show that deletion of components of either the HSP90 or the HSP70 chaperone machinery delays this induction. This delay remains when the galactose-signaling pathway is bypassed, and it cannot be explained by a chaperone requirement for DNA binding by Gal4. Removal of promoter-bound nucleosomes is delayed in a chaperone mutant, and our findings suggest an involvement of HSP90 and HSP70 in this early step in gene induction. [ABSTRACT FROM AUTHOR]

The yeast cyclin-dependent kinase Srb 10 phosphorylates various transcriptional activators as they activate transcription, and acidic transcriptional activating domains found on several activators directly bind Srb 10. Here we show that the interaction between SrblO (with its associated cyclin Srbl1) 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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Eukaryotic DNA is packaged in nucleosomes. How does this sequestration affect the ability of transcription regulators to access their sites? We cite evidence against the idea that nucleosome positioning is determined primarily by the intrinsic propensities of DNA sequences to form nucleosomes – such that, for example, regulatory sites would be ‘nucleosome-free’. Instead, studies in yeast show that nucleosome positioning is primarily determined by specific DNA-binding proteins. Where nucleosomes would otherwise compete with regulatory protein binding (a modest but potentially biologically important effect), this obstacle can be relieved by at least two strategies for exposing regulatory sites. In contrast to their lack of effect on nucleosome positioning, DNA sequence differences do directly affect both the efficiencies with which nucleosomes form in regions flanking regulatory sites before induction, and the extent of their removal upon induction. These nucleosomes, evidently, inhibit basal transcription but are poised to be removed quickly upon command. [ABSTRACT FROM AUTHOR]

Summary: Several transcriptional activators, called “classical” because each bears a natural acidic activating region attached to a DNA binding domain, are proteolytically unstable in yeast, and it has been suggested that this instability is required for transcriptional activation []. Here we test the generality of that proposal by examining a set of activators (called “nonclassical”) that lack activating regions. These activators (e.g., LexA-Gal11) comprise a LexA DNA binding domain fused to a component of the Mediator and are believed to insert the latter into the Mediator and recruit it (and, indirectly, other components required for transcription) to a gene bearing LexA sites []. We find that three, and only three, Mediator subunits, all from its tail domain, work as activators when fused to LexA. All three are unstable, and for the case analyzed in detail, stabilization decreases activity. Thus, to the extent tested, both classical and nonclassical activators work most efficiently when proteolytically unstable. [Copyright &y& Elsevier]

Summary: How is chromatin architecture established and what role does it play in transcription? We show that the yeast regulatory locus UASg bears, in addition to binding sites for the activator Gal4, sites bound by the RSC complex. RSC positions a nucleosome, evidently partially unwound, in a structure that facilitates Gal4 binding to its sites. The complex comprises a barrier that imposes characteristic features of chromatin architecture. In the absence of RSC, ordinary nucleosomes encroach over the UASg and compete with Gal4 for binding. Taken with our previous work, the results show that both prior to and following induction, specific DNA-binding proteins are the predominant determinants of chromatin architecture at the GAL1/10 genes. RSC/nucleosome complexes are also found scattered around the yeast genome. Higher eukaryotic RSC lacks the specific DNA-binding determinants found on yeast RSC, and evidently Gal4 works in those organisms despite whatever obstacle broadly positioned nucleosomes present. [ABSTRACT FROM AUTHOR]

A new nucleosome-occupancy technique reveals how the transcriptional activator Gal4 determines chromatin structure as genes are activated and repressed. [ABSTRACT FROM AUTHOR]

Several yeast activators are phosphorylated by SRB10, a cyclindependent 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. [ABSTRACT FROM AUTHOR]

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 oligoprotine "molecular rulers" in the 18-45 Å length range. We find that there is an optimal linker length which separates the DNA and the activation region for transcription activation. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Examines the role of mediator protein Gal11 as an essential target of P201 nonacidic peptide. Mutagenesis of Gal11; use of the split-ubiquitin system; Effects of Gal11 on different 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. [Copyright &y& Elsevier]

Shows that in Saccharomyces cerevisiae yeast, a gene bearing an enhancer position downstream of the gene is activated if the reporter is linked to a telomere, but not if it is positioned at an internal chromosomal locus. Finding that yeast telomeres form back-folding, or looped, structures; How the findings might explain why transcription of some higher eukaryotic genes depends on their location in heterochromatin.

Presents a study which shows that the Drosophila HMG1-like protein DSP1 and its mammalian homolog hHMG2 interacts with SP100B, a splice variant of SP100A, and that SP100B in turn binds to human homologs of HP1. Ability of DSP1 to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion in yeast; Information on the Drosophila protein.

Examines the transcriptional activation and chromatin remodelling at the PHO5 promoter in yeast by fusion proteins through the recruitment of RNA polymerase II holoenzyme to DNA. Induction of the PHO5 gene in yeast; Usage of gene activation in yeast; Usage of non-classic activators in the experiments.

A barrier phases nucleosomes at the yeast (Saccharomyces cerevisiae) GAL1-GAL10 genes. Here we separate nucleosome positioning from occupancy and show that the degree of occupancy of these phased sites is predictably determined by the underlying DNA sequences. As this occupancy is increased (by sequence alteration), nucleosome removal upon induction is decreased, as is mRNA production. These results explain why promoter sequences have evolved to form nucleosomes relatively inefficiently. [ABSTRACT FROM AUTHOR]