Isogenic strain construction and gene mapping in Candida albicans.

In eukaryotes, the core promoter serves as a platform for the assembly of transcription preinitiation complex (PIC) that includes TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II (pol II), which function collectively to specify the transcription start site. PIC formation usually begins with TFIID binding to the TATA box, initiator, and/or downstream promoter element (DPE) found in most core promoters, followed by the entry of other general transcription factors (GTFs) and pol II through either a sequential assembly or a preassembled pol II holoenzyme pathway. Formation of this promoter-bound complex is sufficient for a basal level of transcription. However, for activator-dependent (or regulated) transcription, general cofactors are often required to transmit regulatory signals between gene-specific activators and the general transcription machinery. Three classes of general cofactors, including TBP-associated factors (TAFs), Mediator, and upstream stimulatory activity (USA)-derived positive cofactors (PC1/PARP-1, PC2, PC3/DNA topoisomerase I, and PC4) and negative cofactor 1 (NC1/HMGB1), normally function independently or in combination to fine-tune the promoter activity in a gene-specific or cell-type-specific manner. In addition, other cofactors, such as TAF1, BTAF1, and negative cofactor 2 (NC2), can also modulate TBP or TFIID binding to the core promoter. In general, these cofactors are capable of repressing basal transcription when activators are absent and stimulating transcription in the presence of activators. Here we review the roles of these cofactors and GTFs, as well as TBP-related factors (TRFs), TAF-containing complexes (TFTC, SAGA, SLIK/SALSA, STAGA, and PRC1) and TAF variants, in pol II-mediated transcription, with emphasis on the events occurring after the chromatin has been remodeled but prior to the formation of the first phosphodiester bond.

The general transcription machinery and general cofactors.

Histone proteins are the major protein components of chromatin, the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells Chromatin is the substrate of many biological processes, such as gene regulation and transcription, replication, mitosis and apoptosis Since histones are extensively post-translationally modified, the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance Many different biochemical techniques have been developed to purify and separate histone proteins Here, we present standard protocols for acid extraction and salt extraction of histones from chromatin; separation of extracted histones by reversed-phase HPLC; analysis of histones and their specific post-translational modification profiles by acid urea (AU) gel electrophoresis and the additional separation of non-canonical histone variants by triton AU(TAU) and 2D TAU electrophoresis; and immunoblotting of isolated histone proteins with modification-specific antibodies

Extraction, purification and analysis of histones.

http://biology.hunter.cuny.edu/molecularbio/Class%20Materials%20Fall%202013%20Biol203/Extra%20Reading/NMD%202009%20Transcription%20Translation.pdf

Pre-mRNA processing reaches back to transcription and ahead to translation.

https://www.cell.com/cell/pdf/S0092-8674(00)80602-9.pdf

Insights into the Function of Rim Protein in Photoreceptors and Etiology of Stargardt's Disease from the Phenotype in abcr Knockout Mice

Summary RNAP II, and its activity is stimulated by the general transcription factor TFIIF and repressed by TFIIB (Kobor Phosphorylation of serine-2 (S2) and serine-5 (S5) of et al., 2000). Mammalian Fcp1 exhibits broad substratethe C-terminal domain (CTD) of RNA polymerase II specificity, dephosphorylating RNAP IIO S2-P and S5-P (RNAP II) is a dynamic process that regulates the tran- with comparable efficiencies in vitro (Lin et al., 2002). scription cycle and coordinates recruitment of RNA However, fission yeast Fcp1 preferentially dephosphor- processing factors. The Fcp1 CTD phosphatase cata- ylates S2-P using synthetic CTD substrates (Hausmann lyzes dephosphorylation of S2-P. Here, we report that andShuman,2002),and fcp1 mutationsinbuddingyeast Ssu72,acomponentoftheyeastcleavage/polyadenyl- enhance preferentially the levels of RNAP IIO S2-P (Cho ationfactor(CPF)complex,is aCTDphosphatasewith et al., 2001a). Recently, a family of small CTD phospha- specificity for S5-P. Ssu72 catalyzes CTD S5-P de-

https://www.cell.com/molecular-cell/pdf/S1097-2765(04)00235-7.pdf

Short Article Ssu72 Is an RNA Polymerase II CTD Phosphatase

Fungal APSES proteins regulate morphogenetic processes, including filamentation and differentiation. The human fungal pathogen Candida albicans contains two APSES proteins: the regulator Efg1p and its homologue Efh1p, described here. Overexpression of EFG1 or EFH1 led to similar phenotypes, including pseudohypha formation and opaque-white switching. An efh1 deletion generated no phenotype under most conditions but caused hyperfilamentation in an efg1 background under embedded or hypoxic conditions. This suggests cooperation of these APSES proteins in the suppression of an alternative morphogenetic signaling pathway. Genome-wide transcriptional profiling revealed that EFG1 and EFH1 regulate partially overlapping sets of genes associated with filament formation. Unexpectedly, Efg1p not only regulates genes involved in morphogenesis but also strongly influences the expression of metabolic genes, inducing glycolytic genes and repressing genes essential for oxidative metabolism. Using one- and two-hybrid assays, we further demonstrate that Efg1p is a repressor, whereas Efh1p is an activator of gene expression. Overall, the results suggest that Efh1p supports the regulatory functions of the primary regulator, Efg1p, and indicate a dual role for these APSES proteins in the regulation of fungal morphogenesis and metabolism.

/pdf/apses-proteins-regulate-morphogenesis-and-metabolism-in-3lgknrmjt6.pdf

APSES Proteins Regulate Morphogenesis and Metabolism in Candida albicans

Ssu72 Is an RNA Polymerase II CTD Phosphatase

TPK1 and TPK2 encode both isoforms of protein kinase A (PKA) catalytic subunits in Candida albicans. Mutants lacking both TPK1 alleles showed defective hyphal morphogenesis on solid inducing media, whereas in liquid hypha, formation was affected slightly. In contrast, tpk2 mutants were only partially morphogenesis defective on solid media, whereas a strong block was observed in liquid. In addition, the yeast forms of tpk2-- but not tpk1-- mutants were completely deficient in invading agar. Because Tpk1p and Tpk2p differ in their N-terminal domains of approximately 80--90 amino acids, while the catalytic portions are highly homologous, the functions of hybrid Tpk proteins with exchanged N-terminal domains were tested. The results demonstrate that the catalytic portions mediate Tpk protein specificities with regard to filamentation, whereas agar invasion is mediated by the N-terminal domain of Tpk2p. Homozygous tpk1 and tpk2 mutants grew normally; however, a tpk2 mutant strain containing a single regulatable TPK1 allele (PCK1p-TPK1) at low expression levels was severely growth defective. It was completely blocked in hyphal morphogenesis and was stress resistant to high osmolarities or temperatures. Thus, both Tpk isoforms in C. albicans share growth functions but, unlike Saccharomyces cerevisiae isoforms, they have positive, specific roles in filament formation in different environments.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2001.02688.x

Distinct and redundant roles of the two protein kinase A isoforms Tpk1p and Tpk2p in morphogenesis and growth of Candida albicans.

DNA loops that juxtapose the promoter and terminator regions of RNA polymerase II-transcribed genes have been identified in yeast and mammalian cells. Loop formation is transcription-dependent and requires components of the pre-mRNA 3′-end processing machinery. Here we report that looping at the yeast GAL10 gene persists following a cycle of transcriptional activation and repression. Moreover, GAL10 and a GAL1p-SEN1 reporter undergo rapid reactivation kinetics following a cycle of activation and repression—a phenomenon defined as “transcriptional memory”—and this effect correlates with the persistence of looping. We propose that gene loops facilitate transcriptional memory in yeast.

/pdf/a-physiological-role-for-gene-loops-in-yeast-n0gmunby0e.pdf

Shankarling Krishnamurthy

Papers

Short Article Ssu72 Is an RNA Polymerase II CTD Phosphatase

APSES Proteins Regulate Morphogenesis and Metabolism in Candida albicans

Ssu72 Is an RNA Polymerase II CTD Phosphatase

Distinct and redundant roles of the two protein kinase A isoforms Tpk1p and Tpk2p in morphogenesis and growth of Candida albicans.

A physiological role for gene loops in yeast