Mary Sopta

Yeast Gal4: a transcriptional paradigm revisited

During the past two decades, the yeast Gal4 protein has been used as a model for studying transcriptional activation in eukaryotes. Many of the properties of transcriptional regulation first demonstrated for Gal4 have since been shown to be reiterated in the function of several other eukaryotic transcriptional regulators. Technological advances based on the transcriptional properties of this factor—such as the two‐hybrid technology and Gal4‐inducible systems for controlled gene expression—have had far‐reaching influences in fields beyond transcription. In this review, we provide an updated account of Gal4 function, including data from new technologies that have been recently applied to the study of the GAL network.

RAP30/74: a general initiation factor that binds to RNA polymerase II.

We have previously shown by affinity chromatography that RAP30 and RAP74 are the mammalian proteins that have the highest affinity for RNA polymerase II. Here we show that RAP30 binds to RAP74 and that the RAP30-RAP74 complex (RAP30/74) is required for accurate initiation by RNA polymerase II. RAP30/74 is required for accurate transcription from the following promoters: the adenovirus major late promoter, the long terminal repeat of human immunodeficiency virus, P2 of the human c-myc gene, the mouse beta maj-globin promoter (all of which have TATA boxes), and the mouse dihydrofolate reductase promoter (which lacks a TATA box). RAP30/74 is not required for initiation by RNA polymerase III at the adenovirus virus-associated RNA promoters. Therefore, RAP30/74 is a general initiation factor that binds to RNA polymerase II.

Telomerase regulation at the crossroads of cell fate

Telomeres are specialized structures at the ends of eukaryotic chromosomes and are crucial for genome stability, cell growth control and carcinogenesis. Normally, they protect chromosomes from end to end fusion, degradation and recombination. Telomerase is a ribonucleoprotein essential for maintenance of telomeres and it is active in germ cells, stem cells and ∼90% of cancers but not in most normal somatic cells. Human telomerase catalytic protein subunit hTERT is crucial for telomerase activity. Although hTERT expression is sufficient to immortalize normal human cells in culture, spontaneous immortalization is extremely rare which suggests that its expression is under strong negative control. Characterization of the hTERT promoter has allowed for the analysis of potential mechanisms of hTERT expression and regulation. The hTERT promoter is very complex and contains a great number of canonical and non-canonical sequences that bind or potentially bind a variety of transcription factors. In this review we focus on the positive and negative regulators of hTERT transcription and their role in normal cell growth and immortalization.

The two faces of Cdk8, a positive/negative regulator of transcription.

Three cyclin dependent kinases, Cdk7, Cdk8 and Cdk9 are intimately connected with the processes of RNA polymerase II dependent transcription initiation and elongation in eukaryotic cells. Each of these kinases is part of a larger multisubunit complex, TFIIH, Mediator and p-TEFb respectively. Of the three kinases, Cdk8 is the most complex given that it has been associated with both positive and negative effects on transcription via mechanisms that include regulation of transcription factor turnover, regulation of CTD phosphorylation and regulation of activator or repressor function. Furthermore, Cdk8 has emerged as a key regulator of multiple transcriptional programs linked to nutrient/growth factor sensing and differentiation control. As such Cdk8 represents a potentially interesting therapeutic drug target. In this review we summarize the current state of knowledge on Cdk8 function both in yeast and higher eukaryotes as well as discussing the effects of Cdk8 null mutations at the organismal level.

Telomeres, Nutrition, and Longevity: Can We Really Navigate Our Aging?

Telomeres are dynamic chromosome-end structures that serve as guardians of genome stability. They are known to be one of the major determinants of aging and longevity in higher mammals. Studies have demonstrated a direct correlation between telomere length and life expectancy, stress, DNA damage, and onset of aging-related diseases. This review discusses the most important factors that influence our telomeres. Various genetic and environmental factors such as diet, physical activity, obesity, and stress are known to influence health and longevity as well as telomere dynamics. Individuals currently have the opportunity to modulate the dynamics of their aging and health span, monitor these processes, and even make future projections by following their telomere dynamics. As telomeres react to positive as well as negative health factors, we should be able to directly influence our telomere metabolism, slow their deterioration, and diminish our aging and perhaps extend our life and health span.

Solvent‐exposed serines in the Gal4 DNA‐binding domain are required for promoter occupancy and transcriptional activation in vivo

The yeast transcriptional activator Gal4 has long been the prototype for studies of eukaryotic transcription. Gal4 is phosphorylated in the DNA-binding domain (DBD); however, the molecular details and functional significance of this remain unknown. We mutagenized seven potential phosphoserines that lie on the solvent-exposed face of the DBD structure and assessed them for transcriptional activity and DNA binding in vivo. Serine to alanine mutants at positions 22, 47, and 85 show the greatest reduction in promoter occupancy and transcriptional activity at the MEL1 promoter containing a single UASGAL . Substitutions with the phosphomimetic aspartate restored DNA-binding and transcriptional activity at serines 22 and 85, suggesting that they are potential sites of Gal4 phosphorylation in vivo. In contrast, the serine to alanine mutants, except serine 22, were fully proficient for binding to the GAL1-10 promoter, containing multiple UASGAL sites, although they had a reduced ability to activate transcription. Collectively, these data show that at the GAL1-10 promoter, functions of the DBD in transcriptional activation can be uncoupled from roles in promoter binding. We suggest that the serines in the DBD mediate protein-protein contacts with the transcription machinery, leading to stabilization of Gal4 at promoters.