RNA Polymerase I Research Articles

The S-Phase Arrest of Host Cells Caused by an Alpha-Herpesvirus Genome Replication Facilitates Viral Recruitment of RNA Polymerase II to Transcribe Viral Genes.

Herpesviruses rely on host RNA polymerae II (RNA Pol II) for their mRNA transcription, yet the mechanisms of which has been poorly defined, while certain herpesviruses can enhance viral gene transcription by altering the RNA Pol II location, modulating its phosphorylation, or directly interacting with RNA Pol II. However, the influence of herpesviruses on RNA Pol II transcription extends beyond these direct effects. Here, we present a novel mechanism by which the host cell cycle regulates viral gene transcription via RNA Pol II during infection by Anatid Herpesvirus 1 (AnHV-1), an avian alpha-herpesvirus. The results demonstrated that the formation of viral replication compartments (vRCs) and the subsequent recruitment of RNA pol II are positively correlated with AnHV-1 DNA synthesis. As viral DNA replication progresses, host cells are arrested in the S phase, which not only halts host gene transcription but also facilitates viral transcription. This cell cycle arrest in the S phase promotes viral DNA (vDNA) synthesis and vRC formation, which further enhances the preferential recruitment of RNA Pol II to viral promoters, enabling efficient viral gene transcription. We propose that this S phase arrest and the hijacking of RNA Pol II represent a novel mechanism by which AnHV-1 enhances viral transcription, offering a unique survival strategy compared to the known strategy in herpesviruses. These findings expand our understanding of herpesvirus-host interactions and highlight potential targets for antiviral strategies.

Research Note: Establishment of vector system harboring duck RNA polymerase I promoter for avian influenza virus

Reverse genetics (RG) systems are extensively utilized to investigate the characteristics of influenza viruses and develop vaccines, predominantly relying on human RNA polymerase I (pol I). However, the efficiency of RG systems for avian-origin influenza viruses may be compromised due to potential species-specific interactions of RNA pol I. In this study, we reported the polymerase activities of the duck RNA pol I promoter in avian cells and the generation of recombinant avian-derived influenza viruses using a novel vector system containing the duck RNA pol I promoter region to enhance the rescue efficiency of the RG system in avian cells. Initially, we explored a putative duck promoter region and identified the optimal size to improve the existing system. Subsequently, we established an RG system incorporating the duck RNA pol I promoter and compared its rescue efficiency with the human pol I system by generating recombinant influenza viruses in several cell lines. Notably, the 250-bp duck RNA pol I promoter demonstrated effective functionality in avian cells, exhibiting higher polymerase activity in a minigenome assay. The newly constructed RG system was significantly improved, enabling the rescue of influenza viruses in 293T, DF-1, and CCL141 cells. Furthermore, HPAI viruses were successfully rescued in DF-1 cells, a result that had not been achieved in previous experiments. In conclusion, our novel RG system harboring duck RNA pol I offers an additional tool for researching influenza viruses and may facilitate the development of vaccines for poultry.

Human Satellite 3 DNA encodes megabase-scale transcription factor binding platforms.

Eukaryotic genomes are frequently littered with large arrays of tandem repeats, called satellite DNA, which underlie the constitutive heterochromatin often found around centromeric regions. While some satellite DNA types have well-established roles in centromere biology, other abundant satellite DNAs have poorly characterized functions. For example, Human Satellite 3 (HSat3), which makes up roughly 2% of the human genome, forms enormous arrays up to tens of megabases, but these arrays play no known roles in centromere function and were almost fully excluded from genome assemblies until recently. As a result, these massive genomic regions have remained relatively understudied, and the potential functional roles of HSat3 have remained largely unknown. To address this, we performed a systematic screen for novel HSat3 binding factors. Our work revealed HSat3 arrays contain high densities of transcription factor (TF) motifs that are bound by factors related to multiple, highly conserved signaling pathways. Unexpectedly, the most enriched TFs in HSat3 belong to the Hippo pathway transcription effector family TEAD. We found that TEAD recruits the co-activator YAP to HSat3 regions in a cell-state specific manner. Using RNA polymerase-I reporter assays, targeted repression of HSat3, inducible degradation of YAP, and super-resolution microscopy, we show that HSat3 arrays can localize YAP/TEAD inside the nucleolus, where YAP regulates RNA Polymerase-I activity. Beyond revealing a direct relationship between the Hippo pathway and ribosomal DNA regulation, this work demonstrates that satellite DNA can encode multiple transcription factor binding motifs, defining a new role for these enormous genomic elements.

Topological stress triggers persistent DNA lesions in ribosomal DNA with ensuing formation of PML-nucleolar compartment.

PML, a multifunctional protein, is crucial for forming PML-nuclear bodies involved in stress responses. Under specific conditions, PML associates with nucleolar caps formed after RNA polymerase I (RNAPI) inhibition, leading to PML-nucleolar associations (PNAs). This study investigates PNAs-inducing stimuli by exposing cells to various genotoxic stresses. We found that the most potent inducers of PNAs introduced topological stress and inhibited RNAPI. Doxorubicin, the most effective compound, induced double-strand breaks (DSBs) in the rDNA locus. PNAs co-localized with damaged rDNA, segregating it from active nucleoli. Cleaving the rDNA locus with I-PpoI confirmed rDNA damage as a genuine stimulus for PNAs. Inhibition of ATM, ATR kinases, and RAD51 reduced I-PpoI-induced PNAs, highlighting the importance of ATM/ATR-dependent nucleolar cap formation and homologous recombination (HR) in their triggering. I-PpoI-induced PNAs co-localized with rDNA DSBs positive for RPA32-pS33 but deficient in RAD51, indicating resected DNA unable to complete HR repair. Our findings suggest that PNAs form in response to persistent rDNA damage within the nucleolar cap, highlighting the interplay between PML/PNAs and rDNA alterations due to topological stress, RNAPI inhibition, and rDNA DSBs destined for HR. Cells with persistent PNAs undergo senescence, suggesting PNAs help avoid rDNA instability, with implications for tumorigenesis and aging.

Honey Targets Ribosome Biogenesis Components to Suppress the Growth of Human Pancreatic Cancer Cells

Pancreatic cancer (PanCa) is one of the deadliest cancers, with limited therapeutic response. Various molecular oncogenic events, including dysregulation of ribosome biogenesis, are linked to the induction, progression, and metastasis of PanCa. Thus, the discovery of new therapies suppressing these oncogenic events and ribosome biogenesis could be a novel therapeutic approach for the prevention and treatment of PanCa. The current study was designed to investigate the anti-cancer effect of honey against PanCa. Our results indicated that honey markedly inhibited the growth and invasive characteristics of pancreatic cancer cells by suppressing the mRNA expression and protein levels of key components of ribosome biogenesis, including RNA Pol-I subunits (RPA194 and RPA135) along with its transcriptional regulators, i.e., UBTF and c-Myc. Honey also induced nucleolar stress in PanCa cells by reducing the expression of various nucleolar proteins (NCL, FBL, and NPM). Honey-mediated regulation on ribosome biogenesis components and nucleolar organization-associated proteins significantly arrested the cell cycle in the G2M phase and induced apoptosis in PanCa cells. These results, for the first time, demonstrated that honey, being a natural remedy, has the potential to induce apoptosis and inhibit the growth and metastatic phenotypes of PanCa by targeting ribosome biogenesis.

Design, synthesis, molecular modeling and evaluation of 2,4-diaminopyrimidine analogues as promising colorectal cancer drugs

The potential of cyclin-dependent kinases (CDKs) as therapeutic targets in cancer treatment is well established. In this study, we present our investigation into a group of 2,4-diaminopyrimidine derivatives that potently inhibit CDK9 and are cytotoxic when tested in colorectal cancer cell lines. We designed and synthesized forty analogues by altering substitutions at C-2 and C-4 position of the pyrimidine system. Among them, compounds 16 h and 16j exhibited strong inhibitory potency against both CDK9 enzymes (IC50 = 11.4 ± 1.4 nM, IC50 = 10.2 ± 1.3 nM respectively) with a significant preference for one over the other, and cytotoxic potency (IC50 = 61 ± 2 nM, IC50 = 20 ± 1 nM respectively) against HCT-116 was discovered through substantial modifications to its structure. Further investigations revealed that compounds 16 h and 16j were directly bound to CDK9, resulting in the suppression of its downstream signaling pathway. This inhibition of cell proliferation occurred by impeding the progression of the cell cycle and inducing apoptosis in cells by suppressing the phosphoryl RNA pol II Ser2. Significantly, compound 16 h and 16j effectively suppressed tumor growth in a xenograft mouse model and exhibited no apparent toxicity. This indicates that CDK9 inhibitors hold great potential as a therapeutic approach for colorectal cancer treatment. Therefore, the aforementioned discoveries are vital for the development of CDK9 inhibitors for the treatment of cancer.

A cyanobacterial sigma factor F controls biofilm-promoting genes through intra- and intercellular pathways

Cyanobacteria frequently constitute integral components of microbial communities known as phototrophic biofilms, which are widespread in various environments. Moreover, assemblages of these organisms, which serve as an expression platform, simplify harvesting the biomass, thereby holding significant industrial relevance. Previous studies of the model cyanobacterium Synechococcus elongatus PCC 7942 revealed that its planktonic growth habit results from a biofilm-suppression mechanism that depends on an extracellular inhibitor, an observation that opens the door to investigating cyanobacterial intercellular communication. Here, we demonstrate that the RNA polymerase sigma factor SigF1, is required for this biofilm-suppression mechanism whereas the S. elongatus paralog SigF2 is not involved in biofilm regulation. Comprehensive transcriptome analyses identified distinct regulons under the control of each of these sigma factors. sigF1 inactivation substantially lowers transcription of genes that code for the primary pilus subunit and consequently prevents pilus assembly. Moreover, additional data demonstrate absence of the biofilm inhibitor from conditioned medium of the sigF1 mutant, further validating involvement of the pilus assembly complex in secretion of the biofilm inhibitor. Consequently, expression is significantly upregulated for the ebfG-operon that encodes matrix components and the genes that encode the corresponding secretion system, which are repressed by the biofilm inhibitor in the wild type. Thus, this study uncovers a basic regulatory component of cyanobacterial intercellular communication, a field that is in its infancy. Elevated expression of biofilm-promoting genes in a sigF1 mutant supports an additional layer of regulation by SigF1 that operates via an intracellular mechanism.

RNA polymerase I mutant affects ribosomal RNA processing and ribosomal DNA stability

ABSTRACT Transcription is a major contributor to genomic instability. The ribosomal RNA (rDNA) gene locus consists of a head-to-tail repeat of the most actively transcribed genes in the genome. RNA polymerase I (RNAPI) is responsible for massive rRNA production, and nascent rRNA is co-transcriptionally assembled with early assembly factors in the yeast nucleolus. In Saccharomyces cerevisiae, a mutant form of RNAPI bearing a fusion of the transcription factor Rrn3 with RNAPI subunit Rpa43 (CARA-RNAPI) has been described previously. Here, we show that the CARA-RNAPI allele results in a novel type of rRNA processing defect, associated with rDNA genomic instability. A fraction of the 35S rRNA produced in CARA-RNAPI mutant escapes processing steps and accumulates. This accumulation is increased in mutants affecting exonucleolytic activities of the exosome complex. CARA-RNAPI is synthetic lethal with monopolin mutants that are known to affect the rDNA condensation. CARA-RNAPI strongly impacts rDNA organization and increases rDNA copy number variation. Reduced rDNA copy number suppresses lethality, suggesting that the chromosome segregation defect is caused by genomic rDNA instability. We conclude that a constitutive association of Rrn3 with transcribing RNAPI results in the accumulation of rRNAs that escape normal processing, impacting rDNA organization and affecting rDNA stability.

Spores of Clostridioides difficile are toxin delivery vehicles

Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells.

Regulators of rDNA array morphology in fission yeast.

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.

TBP facilitates RNA Polymerase I transcription following mitosis

ABSTRACT The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.

Investigating the inhibitory potential of natural bioactive compounds against cyclin-dependent kinase 13: virtual high throughput screening and MD simulation studies to target CDK signaling

Cyclin-dependent kinase 13 (CDK13) belongs to the cyclin-dependent kinase (CDK) family that is actively involved in transcription regulation and RNA splicing. CDK13 binds with its partner, cyclin K, to regulate several biological processes. CDK13 and cyclin K complex phosphorylates RNA pol II carboxyl-terminal domain (CTD) at several serine residues, creating transcription elongation. The upregulation of the kinase contributes to tumor growth and cell proliferation, and is highly associated with various cancers, including skin, stomach, and ovarian. Thus, it can be considered an efficient therapeutic target for the development of drugs against cancer. In this work, a virtual high throughput screening (vHTS) of the ZINC library was carried out to elucidate the initial potent compounds. Further, filters were applied to identify the hit compounds among the ∼90,000 compound library. Based on the docking scores and binding affinity, the top 100 hits were elucidated, and they were further narrowed down to 50 compounds based on ADMET and Lipinski’s RO5 filter. Finally, 10 compounds were chosen that showed appreciable biological activity. Among them, ZINC02136558 was selected as a potent lead compound that showed strong interaction with the amino acid residues of active and binding sites of CDK13. Furthermore, the all-atom molecular dynamic simulation was performed at 200 ns to explore the dynamic evolution of the system. Finally, the results showed that the ZINC02136558 may be considered as a potential lead molecule to inhibit CDK13 and implicated in therapeutic management of cancer and associated diseases.

MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription.

Loss-of-function mutations in methyl-CpG binding protein 2 ( MECP2 ) cause Rett syndrome, a postnatal neurodevelopmental disorder that occurs in ∼1/10,000 live female births. MeCP2 binds to methylated cytosines across genomic DNA and recruits various partners to regulate gene expression. MeCP2 has been shown to repress transcription in vitro and interacts with co-repressors such as the Sin3A and NCoR complexes. Based on these observations, MeCP2 has been largely considered as a repressor of transcription. However, a mouse model of RTT displays many down-regulated genes, and those same genes are up-regulated in a MECP2 duplication mouse model. Furthermore, TCF20, which has been associated with transcriptional activation, have recently been identified as a protein interactor of MeCP2. These data broaden the potential functions of MeCP2 as a regulator of gene expression. Yet, the molecular mechanisms underlying MeCP2-dependent gene regulation remain largely unknown. Here, using a human MECP2 gain-of-function Drosophila model, we screened for genetic modifiers of MECP2 -induced phenotypes. Our approach identified several subunits of the Drosophila super elongation complex, a P-TEFb containing RNA polymerase II (RNA pol II) elongation factor required for the release of promoter-proximally paused RNA pol II, as genetic interactors of MECP2 . We discovered that MeCP2 physically interacts with the SEC in human cells and in the mouse brain. Furthermore, we found that MeCP2 directly binds AFF4, the scaffold of the SEC, via the transcriptional repression domain. Finally, loss of MeCP2 in the mouse cortex caused reduced binding of AFF4 specifically on a subset of genes involved in the regulation of synaptic function, which also displayed the strongest decrease in RNA pol II binding in the genebody. Taken together, our study reveals a previously unrecognized mechanism through which MeCP2 regulates transcription, providing a new dimension to its regulatory role in gene expression.

Integration of molecular docking and molecular dynamics simulations with subtractive proteomics approach to identify the novel drug targets and their inhibitors in Streptococcus gallolyticus

Streptococcus gallolyticus (Sg) is a non-motile, gram-positive bacterium that causes infective endocarditis (inflammation of the heart lining). Because Sg has gained resistance to existing antibiotics and there is currently no drug available, developing effective anti-Sg drugs is critical. This study combined core proteomics with a subtractive proteomics technique to identify potential therapeutic targets for Sg. Several bioinformatics approaches were used to eliminate non-essential and human-specific homologous sequences from the bacterial proteome. Then, virulence, druggability, subcellular localization, and functional analyses were carried out to specify the participation of significant bacterial proteins in various cellular processes. The pathogen’s genome contained three druggable proteins, glucosamine-1phosphate N-acetyltransferase (GlmU), RNA polymerase sigma factor (RpoD), and pantetheine-phosphate adenylyltransferase (PPAT) which could serve as effective targets for developing novel drugs. 3D structures of target protein were modeled through Swiss Model. A natural product library containing 10,000 molecules from the LOTUS database was docked against therapeutic target proteins. Following an evaluation of the docking results using the glide gscore, the top 10 compounds docked against each protein receptor were chosen. LTS001632, LTS0243441, and LTS0236112 were the compounds that exhibited the highest binding affinities against GlmU, PPAT, and RpoD, respectively, among the compounds that were chosen. To augment the docking data, molecular dynamics simulations and MM-GBSA binding free energy were also utilized. More in-vitro research is necessary to transform these possible inhibitors into therapeutic drugs, though computer validations were employed in this study. This combination of computational techniques paves the way for targeted antibiotic development, which addresses the critical need for new therapeutic strategies against S. gallolyticus infections.

Unprocessed snRNAs Are a Prognostic Biomarker and Correlate with a Poorer Prognosis in Colorectal Cancer.

The human Integrator complex is a set of 15 subunits that mediates processing of small nuclear RNAs (snRNAs), and which later participates in splicing messenger RNAs (mRNAs). In addition, it controls the pause and release of RNA polymerase II (RNA pol II) at specific gene promoters in response to growth factors. Mutations in Integrator-complex subunit 6 (INTS6) are associated with different types of tumors. However, the INTS6 gene product does not have a significant prognostic value as a biomarker for tumor progression. Here we show that Integrator-complex deregulation is involved in 8.3% of the colorectal cancer cases diagnosed from the population screen carried out in La Rioja (Spain) from the years 2017 to 2019. Lack of Integrator-complex function, measured by an increased level of unprocessed snRNA, is a prognostic biomarker and correlates with a poorer prognosis in colorectal-cancer patients. The transcriptomic profile of all analyzed colorectal tumors shows a strong alteration of the metabolic state of tumor cells, which compromises standard energy production through mitochondrial respiration, known as the Warburg effect. Furthermore, there is a significant upregulation of genes involved in extracellular matrix organization and collagen rearrangement. This is consistent with tissue reorganization in a growing tumor forming a polyp. Crossing the molecular data generated in this study with the follow-up of patients from population screening indicates that population screening combined with early typing of tumors appears to be the most efficient way to increase patient survival.

Pharmacophore modelling based virtual screening and molecular dynamics identified the novel inhibitors and drug targets against Waddlia chondrophila

Waddlia chondrophila is a possible cause of fetal death in humans. This Chlamydia-related bacterium is an emergent pathogen that causes human miscarriages and ruminant abortions, which results in financial losses. Despite the years of efforts, the underlying mechanism behind the pathogenesis of W. chondrophila is little known which hindered the development of novel treatment options. In the framework of current study, computational approaches were used to identify novel inhibitors (phytocompounds) and drug targets against W. chondrophila. At first, RNA polymerase sigma factor SigA and 3-deoxy-d-manno-octulosonic acid transferase were identified through subtractive proteomics pipeline. Afterwards, extensive docking and simulation analyses were conducted to optimize potentially novel phytocompounds by assessing their binding affinity to target proteins. A 100ns molecular dynamics simulation well complimented the compound's binding affinity and indicated strong stability of predicted compounds at the docked site. The calculation of binding free energies with MMGBSA corroborated the significant binding affinity between phytocompounds and target protein binding sites. The proposed phytocompounds may be a viable treatment option for patients infected with W. chondrophila; however, further research is required to ensure their safety.

ZMYND8 Is a Regulator of Sonic Hedgehog Signaling in ATRA-Mediated Differentiation of Neuroblastoma Cells.

Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8) is a crucial epigenetic regulator that plays a multifaceted role in governing a spectrum of vital cellular processes, encompassing proliferation, apoptosis, migration, tumor suppression, and differentiation. It has emerged as a key player in neuronal differentiation by orchestrating the expression of neuronal lineage-committed genes. The present study uncovers the role of ZMYND8 in regulating the Sonic Hedgehog (SHH) signaling axis, which is crucial for neuronal differentiation. Genetic deletion of ZMYND8 leads to a significant reduction in SHH pathway genes, GLI1, and PTCH1 expression during all-trans-retinoic acid (ATRA)-induced differentiation. ZMYND8 and RNA pol II S5P are found to co-occupy the GLI1 and PTCH1 gene promoters, positively impacting their gene transcription upon ATRA treatment. Interestingly, ZMYND8 is found to counteract the inhibitory effects of Cyclopamine that block the upstream SHH pathway protein SMO, resulting in enhanced neurite formation in neuroblastoma cells following their treatment with ATRA. These results indicate that ZMYND8 is an epigenetic regulator of the SHH signaling pathway and has tremendous therapeutic potential in ATRA-mediated differentiation of neuroblastoma.

Maf1 phosphorylation is regulated through the action of prefoldin-like Bud27 on PP4 phosphatase in Saccharomyces cerevisiae.

Bud27 is a prefoldin-like protein that participates in transcriptional regulation mediated by the three RNA polymerases in Saccharomyces cerevisiae. Lack of Bud27 significantly affects RNA pol III transcription, although the involved mechanisms have not been characterized. Here, we show that Bud27 regulates the phosphorylation state of the RNA pol III transcriptional repressor, Maf1, influences its nuclear localization, and likely its activity. We demonstrate that Bud27 is associated with the Maf1 main phosphatase PP4 in vivo, and that this interaction is required for proper Maf1 dephosphorylation. Lack of Bud27 decreases the interaction among PP4 and Maf1, Maf1 dephosphorylation, and its nuclear entry. Our data uncover a new nuclear function of Bud27, identify PP4 as a novel Bud27 interactor and demonstrate the effect of this prefoldin-like protein on the posttranslational regulation of Maf1. Finally, our data reveal a broader effect of Bud27 on PP4 activity by influencing, at least, the phosphorylation of Rad53.

A simple and unified protocol to purify all seven Escherichia coli RNA polymerase sigma factors

RNA polymerase sigma factors are indispensable in the process of bacterial transcription. They are responsible for a given gene’s promoter region recognition on template DNA and hence determine specificity of RNA polymerase and play a significant role in gene expression regulation. Here, we present a simple and unified protocol for purification of all seven Escherichia coli RNA polymerase sigma factors. In our approach, we took advantage of the His8-SUMO tag, known to increase protein solubilization. Sigma factors were first purified in N-terminal fusions with this tag, which was followed by tag removal with Ulp1 protease. This allowed to obtain proteins in their native form. In addition, the procedure is simple and requires only one resin type. With the general protocol we employed, we were able to successfully purify σD, σE, σS, and σN. Final step modification was required for σF, while for σH and σFecI, denaturing conditions had to be applied. All seven sigma factors were fully functional in forming an active holoenzyme with core RNA polymerase which we demonstrated with EMSA studies.

Determinants of mer Promoter Activity from Pseudomonas aeruginosa.

Since the MerR family is known for its special regulatory mechanism, we aimed to explore which factors determine the expression activity of the mer promoter. The Tn501/Tn21 mer promoter contains an abnormally long spacer (19 bp) between the -35 and -10 elements, which is essential for the unique DNA distortion mechanism. To further understand the role of base sequences in the mer promoter spacer, this study systematically engineered a series of mutant derivatives and used luminescent and fluorescent reporter genes to investigate the expression activity of these derivatives. The results reveal that the expression activity of the mer promoter is synergistically modulated by the spacer length (17 bp is optimal) and the region upstream of -10 (especially -13G). The spacing is regulated by MerR transcription factors through symmetrical sequences, and -13G presumably functions through interaction with the RNA polymerase sigma-70 subunit.