Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I.

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.

RNA polymerase II plays an active role in the formation of gene loops through the Rpb4 subunit.

Gene loops are formed by the interaction of initiation and termination factors occupying the distal ends of a gene during transcription. RNAPII is believed to affect gene looping indirectly owing to its essential role in transcription. The results presented here, however, demonstrate a direct role of RNAPII in gene looping through the Rpb4 subunit. 3C analysis revealed that gene looping is abolished in the rpb4Δ mutant. In contrast to the other looping-defective mutants, rpb4Δ cells do not exhibit a transcription termination defect. RPB4 overexpression, however, rescued the transcription termination and gene looping defect of sua7-1, a mutant of TFIIB. Furthermore, RPB4 overexpression rescued the ssu72-2 gene looping defect, while SSU72 overexpression restored the formation of gene loops in rpb4Δ cells. Interestingly, the interaction of TFIIB with Ssu72 is compromised in rpb4Δ cells. These results suggest that the TFIIB-Ssu72 interaction, which is critical for gene loop formation, is facilitated by Rpb4. We propose that Rpb4 is promoting the transfer of RNAPII from the terminator to the promoter for reinitiation of transcription through TFIIB-Ssu72 mediated gene looping.

The telomeric Cdc13-Stn1-Ten1 complex regulates RNA polymerase II transcription.

Specialized telomeric proteins have an essential role in maintaining genome stability through chromosome end protection and telomere length regulation. In the yeast Saccharomyces cerevisiae, the evolutionary conserved CST complex, composed of the Cdc13, Stn1 and Ten1 proteins, largely contributes to these functions. Here, we report genetic interactions between TEN1 and several genes coding for transcription regulators. Molecular assays confirmed this novel function of Ten1 and further established that it regulates the occupancies of RNA polymerase II and the Spt5 elongation factor within transcribed genes. Since Ten1, but also Cdc13 and Stn1, were found to physically associate with Spt5, we propose that Spt5 represents the target of CST in transcription regulation. Moreover, CST physically associates with Hmo1, previously shown to mediate the architecture of S-phase transcribed genes. The fact that, genome-wide, the promoters of genes down-regulated in the ten1-31 mutant are prefentially bound by Hmo1, leads us to propose a potential role for CST in synchronizing transcription with replication fork progression following head-on collisions.

Structural basis of RNA polymerase I stalling at UV light-induced DNA damage.

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the ribosomal RNA (rRNA) precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes UV-induced lesions. We report the electron cryomicroscopy structure of Pol I in an elongation complex containing a cyclobutane pyrimidine dimer (CPD) at a resolution of 3.6 Å. The structure shows that the lesion induces an early translocation intermediate exhibiting unique features. The bridge helix residue Arg1015 plays a major role in CPD-induced Pol I stalling, as confirmed by mutational analysis. These results, together with biochemical data presented here, reveal the molecular mechanism of Pol I stalling by CPD lesions, which is distinct from Pol II arrest by CPD lesions. Our findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA.

RNA polymerase II phosphorylation and gene looping: new roles for the Rpb4/7 heterodimer in regulating gene expression.

In eukaryotes, cellular RNAs are produced by three nuclear RNA polymerases (RNAPI, II, and III), which are multisubunit complexes. They share structural and functional features, although they are specialized in the synthesis of specific RNAs. RNAPII transcribes the vast majority of cellular RNAs, including mRNAs and a large number of noncoding RNAs. The structure of RNAPII is highly conserved in all eukaryotes, consisting of 12 subunits (Rpb1-12) organized into five structural modules, among which the Rpb4 and Rpb7 subunits form the stalk. Early studies suggested an accessory role for Rpb4, because is required for specific gene transcription pathways. Far from this initial hypothesis, it is now well established that the Rpb4/7 heterodimer plays much wider roles in gene expression regulation. It participates in nuclear and cytosolic processes ranging from transcription to translation and mRNA degradation in a cyclical process. For this reason, Rpb4/7 is considered a coordinator of gene expression. New functions have been added to the list of stalk functions during transcription, which will be reviewed herein: first, a role in the maintenance of proper RNAPII phosphorylation levels, and second, a role in the establishment of a looped gene architecture in actively transcribed genes.

Sub1 contacts the RNA polymerase II stalk to modulate mRNA synthesis.

Biogenesis of messenger RNA is critically influenced by the phosphorylation state of the carboxy-terminal domain (CTD) in the largest RNA polymerase II (RNAPII) subunit. Several kinases and phosphatases are required to maintain proper CTD phosphorylation levels and, additionally, several other proteins modulate them, including Rpb4/7 and Sub1. The Rpb4/7 heterodimer, constituting the RNAPII stalk, promote phosphatase functions and Sub1 globally influences CTD phosphorylation, though its mechanism remains mostly unknown. Here, we show that Sub1 physically interacts with the RNAPII stalk domain, Rpb4/7, likely through its C-terminal region, and associates with Fcp1. While Rpb4 is not required for Sub1 interaction with RNAPII complex, a fully functional heterodimer is required for Sub1 association to promoters. We also demonstrate that a complete CTD is necessary for proper association of Sub1 to chromatin and to the RNAPII. Finally, genetic data show a functional relationship between Sub1 and the RNAPII clamp domain. Altogether, our results indicate that Sub1, Rpb4/7 and Fcp1 interaction modulates CTD phosphorylation. In addition, Sub1 interaction with Rpb4/7 can also modulate transcription start site selection and transcription elongation rate likely by influencing the clamp function.

The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition.

Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/ß domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination.

Sub1/PC4, a multifaceted factor: from transcription to genome stability.

Yeast Sub1 and human PC4, two DNA-binding proteins, were originally identified as transcriptional coactivators with a role during transcription preinitiation/initiation. Indeed, Sub1 is a PIC component, and both PC4 and Sub1 also influence the initiation-elongation transition. Moreover, in the specific case of Sub1, it has been clearly reported that it influences processes downstream during mRNA biogenesis, such as transcription elongation, splicing and termination, and even RNAPII phosphorylation/dephosphorylation. Although Sub1 mechanism of action has been mostly unknown up to date, thanks to the recent finding that Sub1 directly interacts with the RNAPII stalk domain, we can envision how it can modulate so many processes. In addition, Sub1 and PC4 participate in RNAPIII transcription as well, and much additional evidence indicates an evolutionarily conserved role for Sub1 and PC4 in the maintenance of genome stability. In this regard, the most novel function of Sub1 and PC4 has been related to the ability of these proteins to bind G-quadruplex DNA structures that may arise as a consequence of the transcription process.

Atmospheric CO2 enrichment and drought stress modify root exudation of barley.

Rising CO2 concentrations associated with drought stress is likely to influence not only aboveground growth, but also belowground plant processes. Little is known about root exudation being influenced by elements of climate change. Therefore, this study wanted to clarify whether barley root exudation responds to drought and CO2 enrichment and whether this reaction differs between an old and a recently released malting barley cultivar. Barley plants were grown in pots filled with sand in controlled climate chambers at ambient (380 ppm) or elevated (550 ppm) atmospheric [CO2 ] and a normal or reduced water supply. Root exudation patterns were examined at the stem elongation growth stage and when the inflorescences emerged. At both dates, root exudates were analyzed for different compounds such as total free amino acids, proline, potassium, and some phytohormones. Elevated [CO2 ] decreased the concentrations in root exudates of some compounds such as total free amino acids, proline, and abscisic acid. Moreover, reduced water supply increased proline, potassium, electric conductivity, and hormone concentrations. In general, the modern cultivar showed higher concentrations of proline and abscisic acid than the old one, but the cultivars responded differentially under elevated CO2 . Plant developmental stage had also an impact on the root exudation patterns of barley. Generally, we observed significant effects of CO2 enrichment, watering levels, and, to a lesser extent, cultivar on root exudation. However, we did not find any mitigation of the adverse effects of drought by elevated CO2 . Understanding the multitude of relationships within the rhizosphere is an important aspect that has to be taken into consideration in the context of crop performance and carbon balance under conditions of climate change.

Rpb4/7 facilitates RNA polymerase II CTD dephosphorylation.

The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAPII) participate in a variety of processes from transcription, DNA repair, mRNA export and decay, to translation regulation and stress response. However, their mechanism(s) of action remains unclear. Here, we show that the Rpb4/7 heterodimer in Saccharomyces cerevisiae plays a key role in controlling phosphorylation of the carboxy terminal domain (CTD) of the Rpb1 subunit of RNAPII. Proper phosphorylation of the CTD is critical for the synthesis and processing of RNAPII transcripts. Deletion of RPB4, and mutations that disrupt the integrity of Rpb4/7 or its recruitment to the RNAPII complex, increased phosphorylation of Ser2, Ser5, Ser7 and Thr4 within the CTD. RPB4 interacted genetically with genes encoding CTD phosphatases (SSU72, FCP1), CTD kinases (KIN28, CTK1, SRB10) and a prolyl isomerase that targets the CTD (ESS1). We show that Rpb4 is important for Ssu72 and Fcp1 phosphatases association, recruitment and/or accessibility to the CTD, and that this correlates strongly with Ser5P and Ser2P levels, respectively. Our data also suggest that Fcp1 is the Thr4P phosphatase in yeast. Based on these and other results, we suggest a model in which Rpb4/7 helps recruit and potentially stimulate the activity of CTD-modifying enzymes, a role that is central to RNAPII function.

The Role of S. cerevisiae Sub1/PC4 in Transcription Elongation Depends on the C-Terminal Region and Is Independent of the ssDNA Binding Domain.

Saccharomyces cerevisiae Sub1 (ScSub1) has been defined as a transcriptional stimulatory protein due to its homology to the ssDNA binding domain (ssDBD) of human PC4 (hPC4). Recently, PC4/Sub1 orthologues have been elucidated in eukaryotes, prokaryotes, and bacteriophages with functions related to DNA metabolism. Additionally, ScSub1 contains a unique carboxyl-terminal region (CT) of unknown function up to date. Specifically, it has been shown that Sub1 is required for transcription activation, as well as other processes, throughout the transcription cycle. Despite the progress that has been made in understanding the mechanism underlying Sub1's functions, some questions remain unanswered. As a case in point: whether Sub1's roles in initiation and elongation are differentially predicated on distinct regions of the protein or how Sub1's functions are regulated. Here, we uncover some residues that are key for DNA-ScSub1 interaction in vivo, localized in the ssDBD, and required for Sub1 recruitment to promoters. Furthermore, using an array of genetic and molecular techniques, we demonstrate that the CT region is required for transcription elongation by RNA polymerase II (RNAPII). Altogether, our data indicate that Sub1 plays a dual role during transcription-in initiation through the ssDBD and in elongation through the CT region.

Structural basis of Nrd1-Nab3 heterodimerization.

Heterodimerization of RNA binding proteins Nrd1 and Nab3 is essential to communicate the RNA recognition in the nascent transcript with the Nrd1 recognition of the Ser5-phosphorylated Rbp1 C-terminal domain in RNA polymerase II. The structure of a Nrd1-Nab3 chimera reveals the basis of heterodimerization, filling a missing gap in knowledge of this system. The free form of the Nrd1 interaction domain of Nab3 (NRID) forms a multi-state three-helix bundle that is clamped in a single conformation upon complex formation with the Nab3 interaction domain of Nrd1 (NAID). The latter domain forms two long helices that wrap around NRID, resulting in an extensive protein-protein interface that would explain the highly favorable free energy of heterodimerization. Mutagenesis of some conserved hydrophobic residues involved in the heterodimerization leads to temperature-sensitive phenotypes, revealing the importance of this interaction in yeast cell fitness. The Nrd1-Nab3 structure resembles the previously reported Rna14/Rna15 heterodimer structure, which is part of the poly(A)-dependent termination pathway, suggesting that both machineries use similar structural solutions despite they share little sequence homology and are potentially evolutionary divergent.

Regulation of Eukaryotic RNAPs Activities by Phosphorylation.

Evolutionarily conserved kinases and phosphatases regulate RNA polymerase II (RNAPII) transcript synthesis by modifying the phosphorylation status of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNAPII. Proper levels of Rpb1-CTD phosphorylation are required for RNA co-transcriptional processing and to coordinate transcription with other nuclear processes, such as chromatin remodeling and histone modification. Whether other RNAPII subunits are phosphorylated and influences their role in gene expression is still an unanswered question. Much less is known about RNAPI and RNAPIII phosphorylation, whose subunits do not contain functional CTDs. However, diverse studies have reported that several RNAPI and RNAPIII subunits are susceptible to phosphorylation. Some of these phosphorylation sites are distributed within subunits common to all three RNAPs whereas others are only shared between RNAPI and RNAPIII. This suggests that the activities of all RNAPs might be finely modulated by phosphorylation events and raises the idea of a tight coordination between the three RNAPs. Supporting this view, the transcription by all RNAPs is regulated by signaling pathways that sense different environmental cues to adapt a global RNA transcriptional response. This review focuses on how the phosphorylation of RNAPs might regulate their function and we comment on the regulation by phosphorylation of some key transcription factors in the case of RNAPI and RNAPIII. Finally, we discuss the existence of possible common mechanisms that could coordinate their activities.

Assessing potential aquatic toxicity of airport runoff using physicochemical parameters and Lemna gibba and Aliivibrio fischeri bioassays.

A critical problem derived from airport operations is the environmental impact of runoff water. Airport runoff includes a complex mixture of pollutants, e.g., from deicing agents, that may affect negatively natural water bodies. This study assesses the spatial and temporal aquatic ecotoxicity of runoff water and possible aeroplane drift in a German airport. Over winter 2012-2013, from November to May, water samples were collected within the airport and surrounding area. These samples were analyzed using traditional physicochemical analysis and biotests with two aquatic organisms from different trophic levels, Lemna gibba and Aliivibrio fischeri. Overall, the samples examined in this study were relatively non-toxic to the tested organisms. The physicochemical parameters were mainly influenced by the sampling period being higher in colder months. In contrast, the ecotoxicity was influenced by the sampling site. For sites within the airport, a high correlation between the physicochemical parameters (EC and TOC) and toxicity in L. gibba was found. These correlations were not evident in samples taken outside the airport or when A. fischeri was used as a bioindicator. However, a pronounced seasonality has been observed, linked to the coldest months with average inhibition values of 50% in L. gibba and 25% in A. fischeri, particularly in January. Both biotests yielded differing results; therefore, more biotests should be included. However, L. gibba showed a good response with this type of water samples to be included in future studies together with detailed chemical analysis. The present study provides data to assess the potential ecotoxicological effects of airport runoff affected by winter operations.

Sub1 and RNAPII, until termination does them part.

Sub1 was initially identified as a coactivator factor with a role during transcription initiation. However, over the last years, many evidences showed that it influences processes downstream during mRNA biogenesis, such as elongation, termination, and RNAPII phosphorylation. The recent discover that Sub1 directly interacts with the RNAPII stalk adds new insights into how it achieves all these tasks.

The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription.

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

Sub1 associates with Spt5 and influences RNA polymerase II transcription elongation rate.

The transcriptional coactivator Sub1 has been implicated in several steps of mRNA metabolism in yeast, such as the activation of transcription, termination, and 3'-end formation. In addition, Sub1 globally regulates RNA polymerase II phosphorylation, and most recently it has been shown that it is a functional component of the preinitiation complex. Here we present evidence that Sub1 plays a significant role in transcription elongation by RNA polymerase II (RNAPII). We show that SUB1 genetically interacts with the gene encoding the elongation factor Spt5, that Sub1 influences Spt5 phosphorylation of the carboxy-terminal domain of RNAPII largest subunit by the kinase Bur1, and that both Sub1 and Spt5 copurify in the same complex, likely during early transcription elongation. Indeed, our data indicate that Sub1 influences Spt5-Rpb1 interaction. In addition, biochemical and molecular data show that Sub1 influences transcription elongation of constitutive and inducible genes and associates with coding regions in a transcription-dependent manner. Taken together, our results indicate that Sub1 associates with Spt5 and influences Spt5-Rpb1 complex levels and consequently transcription elongation rate.

The conserved foot domain of RNA pol II associates with proteins involved in transcriptional initiation and/or early elongation.

RNA polymerase (pol) II establishes many protein-protein interactions with transcriptional regulators to coordinate different steps of transcription. Although some of these interactions have been well described, little is known about the existence of RNA pol II regions involved in contact with transcriptional regulators. We hypothesize that conserved regions on the surface of RNA pol II contact transcriptional regulators. We identified such an RNA pol II conserved region that includes the majority of the "foot" domain and identified interactions of this region with Mvp1, a protein required for sorting proteins to the vacuole, and Spo14, a phospholipase D. Deletion of MVP1 and SPO14 affects the transcription of their target genes and increases phosphorylation of Ser5 in the carboxy-terminal domain (CTD). Genetic, phenotypic, and functional analyses point to a role for these proteins in transcriptional initiation and/or early elongation, consistent with their genetic interactions with CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme.