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The histone acetyltransferase (HAT) subunit of coactivator complex SAGA, Gcn5, stimulates eviction of promoter nucleosomes at certain highly expressed yeast genes, including those activated by transcription factor Gcn4 in amino acid-deprived cells; however, the importance of other HAT complexes in this process was poorly understood. Analyzing mutations that disrupt the integrity or activity of HAT complexes NuA4 or NuA3, or HAT Rtt109, revealed that only NuA4 acts on par with Gcn5, and functions additively, in evicting and repositioning promoter nucleosomes and stimulating transcription of starvation-induced genes. NuA4 is generally more important than Gcn5, however, in promoter nucleosome eviction, TBP recruitment, and transcription at most other genes expressed constitutively. NuA4 also predominates over Gcn5 in stimulating TBP recruitment and transcription of genes categorized as principally dependent on the cofactor TFIID versus SAGA, except for the most highly expressed subset including ribosomal protein genes, where Gcn5 contributes strongly to PIC assembly and transcription. Both SAGA and NuA4 are recruited to promoter regions of starvation-induced genes in a manner that might be feedback controlled by their HAT activities. Our findings reveal an intricate interplay between these two HATs in nucleosome eviction, PIC assembly, and transcription that differs between the starvation-induced and basal transcriptomes.

The agglomeration of magnetic nanoparticles can have a negative effect on the microwave absorption performance. However, the novel nickel-ion-polydopamine complex coating strategy can avoid this occurrence. Hence, ultrafine nickel nanoparticles (Ni NPs) with uniform size loaded on the surface of hollow N-doped carbon spheres (HNC€Ni) have been fabricated by a facile method. The HNC€Ni composites with different amount of nickel ions are investigated. Interestingly, the introduction of nickel ions has three advantages: (1) Decreases the rate that dopamine self-polymerizes into polydopamine, thus avoiding the serious agglomeration. (2) Improves the thermal stability and maintain the original shape without destroying. (3) Acts as a catalyst to enhance the degree of graphitization. Because of the increase of interfacial polarization and conduction loss, the HNC€Ni composites with only 10 wt.% filler loading possess better microwave absorption performance compared to HNC (without adding nickel salt), covering the whole Ku band and the entire X band. Surprisingly, the broadest effective absorption bandwidth (EAB) for three samples of HNC€Ni composites are all 6.8 GHz (11.2–18 GHz). The minimum reflection loss (RLmin) reaches −55.15 dB for HNC€Ni-1 and −62.96 dB for HNC€Ni-2. We believe that this work provides a high-production and low-cost route to synthesize advanced microwave absorption absorbers.

Metal-organic framework (MOF) derivatives show potential as electromagnetic microwave (EMW) absorbers. In this study, a series of MOF-Co@CNTs were prepared by pyrolyzing a ZIF-67/g-C3N4 precursor. ZIF-67 catalyzed the polymerization of g-C3N4 into CNTs on the surface of ZIF-67. The effect of the carbonization temperature and ratio of g-C3N4 to ZIF-67 on the EMW absorption capacity of MOF-Co@CNTs is discussed. The complex permittivity increased upon increasing the temperature, but the complex permeability changed only subtly. The optimal process used a carbonization temperature of 700 °C and a mass ratio of g-C3N4 and Co(NO3)2‧6H2O of 1. The RLmin was −59.5 dB at a thickness of 1.6 mm, and the effective bandwidth (RL

Zeolitic Imidazolate Frameworks (ZIFs) derivative have attracted much attention due to their excellent performance in electromagnetic wave (EMW) absorption. In this work, we designed and synthesized anemone-shape ZIF-67@CNTs by catalyzing melamine (C3N6H6) into carbon nanotubes (CNTs) on the surface of ZIF-67(Co2+ coordinated by imidazole ester). Furthermore, the effects of the amount of melamine added and annealing temperature on ZIF-67@CNTs and its capacity in EMW absorption were discussed. By comparing the comprehensive capacity, it is determined that ZIF-67@CNTs-5(700) (mass ratio: ZIF-67: C3N6H6 = 1:5, annealing temperature: 700 °C) shows the best performance. For ZIF-67@CNTs-5(700) with 10% filled, the maximum effective bandwidth (RL

The rational design on the morphology and microstructure of magnetic carbon-based composites can enhance microwave absorption performance. Herein, a novel hierarchical CoNi@NC hollow microspheres have been successfully synthesized, involving the fabrication of hierarchical CoNi-based carbonate hydroxide hollow microspheres self-assembled by nanosheets using CoNi-glycerate microspheres as self-template, the uniform coating of a layer of polydopamine (PDA) on the surface, and the calcination process at different temperatures. Importantly, the calcination temperature can significantly influence the morphology, microstructures, and microwave absorption performance. All CoNi-based carbonate hydroxide nanosheets coated with PDA can be successfully converted into abundant small CoNi nanoparticles embedded into carbon layer without the agglomeration of CoNi alloys when the calcination temperature is 700 °C. The CoNi@NC-700 sample exhibits outstanding microwave absorption ability especially in X band thanks to optimized impedance matching and enhanced attenuation ability. The RLmin can reach − 69.58 dB at 10.09 GHz with a matching thickness of 2.99 mm, and the effective absorption bandwidth (EAB) is 4.5 GHz (7.97–12.47 GHz) at 3.08 mm, covering the whole X band. Moreover, the broadest EAB is 5.3 GHz (12.7–18 GHz) at 2.13 mm. We believe that this research will provide a new thought to enhance the microwave absorption capability.

Several composites of modified graphitic carbon nitride (MCN) and Fe3O4 particles were obtained through pyrolysing MCN/iron acetylacetone (Fe(acac)3) at different temperatures to form electromagnetic wave (EWM) absorbers. By decreasing the nitrogen content of g-C3N4 and introducing Fe3O4 particles, the EMW absorption ability of the g-C3N4 derivatives (MCN/Fe3O4) improved remarkably. For MCN/Fe3O4-500 with 6 wt% filling, the minimum reflection loss (RLmin) reached −73.93 dB at a thickness of 3.4 mm and the effective absorption bandwidth (EAB) was as high as 5.44 GHz at a thickness of 1.9 mm. Additionally, the mass ratio of MCN and Fe3O4 and the EMW absorption mechanism of MCN/Fe3O4-500 were also discussed. This study provides a new approach for the preparation of high-efficiency EMW absorbers by modification of g-C3N4.

Introduction: Chongqing is among the areas with the highest rubella incidence rates in China. This study aimed to analyze the temporal distribution characteristics of rubella and establish a forecasting model in Chongqing, which could provide a tool for decision-making in the early warning system for the health sector. Methodology: The rubella monthly incidence data from 2004 to 2019 were obtained from the Chongqing Center of Disease and Control. The incidence from 2004 to June 2019 was fitted using the seasonal autoregressive integrated moving average (SARIMA) model and the back-propagation neural network (BPNN) model, and the data from July to December 2019 was used for validation. Results: A total of 30,083 rubella cases were reported in this study, with a significantly higher average annual incidence before the nationwide introduction of rubella-containing vaccine (RCV). The peak of rubella notification was from April to June annually. Both SARIMA and BPNN models were capable of predicting the expected incidence of rubella. However, the linear SARIMA model fits and predicts better than the nonlinear BPNN model. Conclusions: Based on the results, rubella incidence in Chongqing has an obvious seasonal trend, and SARIMA (2,1,1) × (1,1,1) 12 model can predict the incidence of rubella well. The SARIMA model is a feasible tool for producing reliable rubella forecasts in Chongqing.

An environmentally friendly solvent-free strategy for synthesis of velutipes-shaped Ni@CNTs-x composites as high-performance electromagnetic wave (EMW) absorbers by combining Ni particles with carbon nanotubes (Ni@CNTs) through in situ pyrolysis of a mixture of nickel nitrate and melamine is proposed. The CNTs contained several microporous structures, thereby facilitating a reduction in density and an enhancement of the EMW absorption. The EMW absorption performance first increased then decreased with increasing Ni content. The effective absorption bandwidth (RL < −10 dB) of the synthesized Ni@CNTs-x was up to 5.45 GHz for a thickness of 1.79 mm, while the minimum reflection loss (RLmin) was −60.56 dB at 17.32 GHz for a thickness of 1.582 mm and 20 wt.% loading. Overall, the results of this study indicate that this facile environmentally friendly method is effective to prepare excellent, thin, lightweight EMW absorbers with wide absorption bandwidth and high absorption efficiency.

A two-step method has been adopted to synthesize cobalt nanoclusters in carbon (CoC)-decorated reduced graphene oxide (rGO) from graphene oxide (GO) and ZIF-67 as an excellent stable electromagnetic wave (EMW) absorber. Firstly, the electrostatic force between GO and Co2+ gave rise to in situ growth of ZIF-67 on GO (ZIF-67–GO). Then CoC–rGO was obtained by calcination of ZIF-67–GO hybrids. The ligand is introduced as the nitrogen source to GO. The crystal structure, chemical composition, and magnetic properties of CoC–rGO were investigated by x-ray powder diffraction analysis, Fourier-transform infrared (FT-IR) spectroscopy, Raman spectroscopy, x-ray photoelectron spectroscopy, and vibrating-sample magnetometry. The morphology was observed by scanning electron microscopy and transmission electron microscopy. By adjusting the ratio of GO to ZIF-67, the electromagnetic parameters of CoC–rGO can be optimized. It was found that the sample consisting of 10 wt.% CoC–rGO-2 in a paraffin matrix exhibited excellent EMW absorption performance, reaching a minimum reflection loss (RLmin) of −44.77 dB with thickness of 2.1 mm and an effective bandwidth (RL ≤ −10 dB) of up to 5.2 GHz at thickness of 1.8 mm. The results of this study open an effective and simple avenue for the design of EMW absorbers using metal–organic framework (MOF) and low-cost carbon materials.

High-performance and corrosion-resistant microwave absorbers are required response to environmental degradation. Herein, monodisperse and superparamagnetic porous RGO@Fe3O4 nanocomposites are prepared by a simple one-pot method based on solvothermal treatment of Fe(acac)3 and graphene oxide solution. The as-prepared samples have been characterized using XRD, Raman spectroscopy, XPS, SEM and TEM. The magnetic properties and electromagnetic parameters analyses indicated that the nanocomposites show superparamagnetism with relatively high saturation magnetization and excellent electromagnetic wave (EMW) absorption properties. The minimum reflection loss (RLmin) and maximum effective absorption bandwidth (EAB) are − 65.7 dB and 6.0 GHz, respectively. Significantly, after hydrochloric acid treatment for 1 month, RGO@Fe3O4 hybrids still exhibited superior microwave absorption capabilities. It is suggested that our approach proposes an economic and green route for the controllable synthesis of porous functionalized graphene materials, a kind of corrosion-resistant synergistic microwave absorbers.

A unique three-dimensional (3D) structure nanocomposites composed of 3D graphene and shuttle-shaped ZnO nanoparticles (3DGZ) were fabricated via a novel and facile two-step synthetic method in a solution phase technique. The microwave absorption properties of 3DGZ nanocomposites with different ZnO proportions were investigated. Morphology analysis showed that the shuttle-shaped ZnO nanoparticles were uniformly scattered on and embedded into 3D graphene without any aggregation, which is favorable to enhance the interfacial polarization. The sample 3DGZ-2 displayed the most outstanding microwave absorption performance. A minimum reflection loss of −48.05 dB was obtained at 11.71 GHz with a thickness of 1.82 mm, and the RL exceeding −10 dB were acquired in the frequency range covering from 12.98 to 17.06 GHz at a given thickness of 1.5 mm. Considering the outstanding performance of 3DGZ nanocomposites, we believe it has great potential to be used as a highly effective, lightweight, excellent heat stable and tunable microwave absorber.

Background Mumps is classified as a class C infection disease in China, and the Chongqing area has one of the highest incidence rates in the country. We aimed to establish a prediction model for mumps in Chongqing and analyze its seasonality, which is important for risk analysis and allocation of resources in the health sector. Methods Data on incidence of mumps from January 2004 to December 2018 were obtained from Chongqing Municipal Bureau of Disease Control and Prevention. The incidence of mumps from 2004 to 2017 was fitted using a seasonal autoregressive comprehensive moving average (SARIMA) model. The root mean square error (RMSE) and mean absolute percentage error (MAPE) were used to compare the goodness of fit of the models. The 2018 incidence data were used for validation. Results From 2004 to 2018, a total of 159,181 cases (93,655 males and 65,526 females) of mumps were reported in Chongqing, with significantly more men than women. The age group of 0–19 years old accounted for 92.41% of all reported cases, and students made up the largest proportion (62.83%), followed by scattered children and children in kindergarten. The SARIMA(2, 1, 1) × (0, 1, 1)12 was the best fit model, RMSE and MAPE were 0.9950 and 39.8396%, respectively. Conclusion Based on the study findings, the incidence of mumps in Chongqing has an obvious seasonal trend, and SARIMA(2, 1, 1) × (0, 1, 1)12 model can also predict the incidence of mumps well. The SARIMA model of time series analysis is a feasible and simple method for predicting mumps in Chongqing.

The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the −1 and +1 nucleosomes flanking NDRs; however, Ino80C’s function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the −1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.

Dielectric composite shows potential in the field of electromagnetic wave (EMW) absorption. Hence, we designed and synthesized ZnO/C@PPy, by calcining ZIF-8 and coating polypyrrole. It was studied emphatically the effects of calcination temperature and the amount of pyrrole coating on the EMW absorption capacity and permittivity of ZnO/C@PPy. With the increase of temperature and amount of pyrrole and its permittivity enhance significantly. Z700@P4 (ZIF-8 was calcined at 700 0C and pyrrole amount was 0.4 mL/0.1 g ZnO/C) shows the optimal performace with minimum reflection loss (RLmin) of −76.31 dB at 14.85 GHz and the effective absorption bandwidth (RL

Acute haemorrhagic conjunctivitis is a highly contagious eye disease, the prediction of acute haemorrhagic conjunctivitis is very important to prevent and grasp its development trend. We use the exponential smoothing model and the seasonal autoregressive integrated moving average (SARIMA) model to analyse and predict. The monthly incidence data from 2004 to 2017 were used to fit two models, the actual incidence of acute haemorrhagic conjunctivitis in 2018 was used to validate the model. Finally, the prediction effect of exponential smoothing is best, the mean square error and the mean absolute percentage error were 0.0152 and 0.1871, respectively. In addition, the incidence of acute haemorrhagic conjunctivitis in Chongqing had a seasonal trend characteristic, with the peak period from June to September each year.

Although immunization against varicella using vaccines has been proven to be significant and effective in the past decades, varicella remains a major public health concern for many developing countries. Varicella vaccination has not been introduced into routine immunization programs in China, and varicella outbreaks have continued to occur. Taking the city of Chongqing, which has a high prevalence of varicella, as an example, this study explored the spatiotemporal epidemiology of varicella. Based on the reported data of varicella cases from 1 January 2014 to 31 December 2018 in Chongqing, hot spots and space-time clusters of varicella were identified using spatial autocorrelation analysis and scan statistics. Within this period, a total of 112,273 varicella cases were reported in Chongqing (average annual incidence: 73.44 per 100,000), including one death. The incidence of varicella showed an increasing trend with significant seasonal peaks, which occurred during April to July and October to January of the following year. The total ratio of male to female patients affected was 1.10:1. Children under the age of 15 and students accounted for the majority of the patient population. The hotspots detected through local spatial autocorrelation analysis, and the most likely clusters identified by scan analysis, were primarily in the main urban districts of Chongqing. The secondary clusters were mostly detected in northeast and southwest Chongqing. There were obvious spatial dependence and spatiotemporal clustering characteristics of varicella in Chongqing from 2014 to 2018. High-risk districts, populations, and peak periods were found in this study, which could be helpful in implementing varicella prevention and control programs, and in adjusting vaccination strategies for the varicella vaccine based on actual conditions.

Popcorn-like α-Fe2O3/3D graphene sponge (α-F/3DGS) composites have been successfully fabricated by a novel and facile two-step synthetic route combining template-assisted preparation and co-precipitation method for microwave absorption application. The results show that the popcorn-like α-Fe2O3 nanoparticles with an average diameter about 150–200 nm are uniformly encapsulated into the 3DGS exhibiting a porous microstructure with plenty of wrinkles. The composite displays the optimum microwave absorption properties with the minimum reflection loss (RLmin) of − 55.7 dB and the maximum effective microwave absorption bandwidth up to 4.42 GHz (12.30–16.72 GHz) when the thicknesses are merely 1.4 and 1.7 mm, respectively, exhibiting greatly enhanced microwave absorption properties compared to those of some 2D graphene-based microwave absorbing materials. The excellent microwave absorption properties are attributed to the synergistic effect between dielectric loss and magnetic loss mechanism and improvement for the impedance matching behavior. This study paves a way for designing and fabricating lightweight and high performance microwave absorption materials based on 3D graphene composites.

The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of −1 and +1 nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by transcription factor Gcn4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of Gcn4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs—highly depleted of nucleosomes—during transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.

Single-crystal Si, which is one of the best infrared optics materials and an important semiconductor substrate, is difficult to process owing to its inherent brittleness and hardness. Micro-laser-assisted machining (μ-LAM) has the potential for improving surface integrity by combining the advantages of LAM and conventional ultra-precision machining. However, its material removal mechanism differs from that of conventional machining (CM) technology owing to the interaction between LAM and the diamond turning action. In this study, a three-dimensional transient thermal model based on the finite element method was developed to investigate the temperature responses under different machining conditions. To examine the material removal mechanism and feasibility of high-efficiency machining with μ-LAM, the machining parameters, including laser power and rotation speed, were considered. Furthermore, the surface integrity was systematically characterised and analysed. It was observed that the dislocation motion velocity increased to approximately 2.57 × 109 times that of CM, thereby facilitating ductile deformation of single-crystal Si in μ-LAM. Based on the X-ray diffraction (XRD) pattern analysis, the residual stress significantly decreased with μ-LAM. Furthermore, it was revealed that increasing the rotation speed facilitated ductile mode machining and suppressed the occurrence of micro-pits, and the surface roughness and amorphous layer thickness decreased with increasing rotation speed. Thus, this study provides an important reference for μ-LAM of hard and brittle materials.

Rational design of microstructure and components provides an effective method to address the challenge of developing advanced microwave absorption (MA) materials. In this work, combining the preparation of the precursors (SiO2@RF) for the deposition of Fe-glycerate (Fe-G) nanosheets and in situ pyrolysis process, followed by removal of the SiO2 template, the bird-nest-patterned C@Fe3O4 (BN-HDCF) composites with hollow dual-shell structure are rationally constructed to obtain superior MA performance. The results demonstrate that the eddy effect is suppressed by lamellar Fe3O4 shell, which benefits the entrance of electromagnetic waves (EMWs) and the cavity and groove of bowl like carbon core could boost the attenuation of EMWs. Importantly, due to the integration of the synergistic effects and the multiple polarization loss as well as the improved impedance matching, the as-prepared BN-HDCF composites demonstrate excellent MA performance in view of both minimum reflection loss (RLmin, −80 dB) and effective absorption bandwidth (EAB, below −10 dB, 6.1 GHz) with a thickness of 2.23 mm, covering the full X band with a thickness of 3.1 mm. Such impressive MA performance lights the way to the fabrication of the hierarchical structure of advanced absorbents.

A novel ternary core-shell structured ZnOC@CoC@PAN as an electromagnetic wave (EMW) absorber was successfully fabricated by calcining precursor ZIF-8@ZIF-67 and covering the product with conductive polyaniline (PAN). The multicomponent core-shell structure and the conductive polymer coating were conducive to increasing the dielectric loss, magnetic loss, interfacial polarization, and conductivity loss to dissipate EMW energy. Adjusting the amount of aniline gave ZnOC@CoC@PAN-0.5, which had excellent EMW absorption performance. The minimum reflection loss (RLmin) was −50.62 dB at 13.66 GHz and the effective absorption bandwidth (RL

Chaperones, nucleosome remodeling complexes, and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these cofactors function ubiquitously, as well as the impact of nucleosome eviction on transcription genome-wide, is poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple cofactors to address these issues for about 200 genes belonging to the Gcn4 transcriptome, of which about 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 cochaperone Ydj1, and chromatin-associated factor Yta7 are required downstream from Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC, and H2AZ are dispensable for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double, and triple mutants implicated Gcn5, Snf2, and Ydj1 in H3 eviction at most, but not all, Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these three cofactors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in cofactor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes but that other steps in gene activation are more rate-limiting for most other yeast genes.

Grafting modification is an effective means to improve the interfacial bonding properties of fibre composites. In this study, a series of grafted poly(p-phenylene benzobisoxazole)-polyurethane (PBO-PU) fibres were fabricated using online continuous air plasma treatment. The resulting PBO-PU fibres were characterised using attenuated total reflection Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and scanning electron microscopy. The results of interlaminar shear strength (ILSS) tests show that all the PBO-PU fibre reinforced epoxy resin composites exhibited superior interfacial adhesion properties compared with the untreated composites, and the ILSS reached 48.0 MPa, indicating an improvement of 99.49 %. Our findings demonstrate that plasma grafting provides the possibility to realise industrial production of PBO/epoxy composites exhibiting high interlaminar shear strengths.

Gcn4 is a yeast transcriptional activator induced by amino acid starvation. ChIP-seq analysis revealed 546 genomic sites occupied by Gcn4 in starved cells, representing ∼30% of Gcn4-binding motifs. Surprisingly, only ∼40% of the bound sites are in promoters, of which only ∼60% activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining ∼300 Gcn4-bound sites are within coding sequences (CDSs), with ∼75 representing the only bound sites near Gcn4-induced genes. Many such unconventional sites map between divergent antisense and sub-genic sense transcripts induced within CDSs adjacent to induced TBP peaks, consistent with Gcn4 activation of cryptic bidirectional internal promoters. Mutational analysis confirms that Gcn4 sites within CDSs can activate sub-genic and full-length transcripts from the same or adjacent genes, showing that functional Gcn4 binding is not confined to promoters. Our results show that internal promoters can be regulated by an activator that functions at conventional 5'-positioned promoters.

Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment.

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor (GEF) for eukaryotic translation initiation factor 2, which stimulates formation of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) in a manner inhibited by phosphorylated eIF2 [eIF2(αP)]. While eIF2B contains five subunits, the ε/Gcd6 subunit is sufficient for GEF activity in vitro. The δ/Gcd2 and β/Gcd7 subunits function with α/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(αP)-GDP, but the essential functions of δ/Gcd2 and β/Gcd7 are not well understood. We show that the depletion of wild-type β/Gcd7, three lethal β/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in β/Gcd7 and eIF2α all impair eIF2 binding to eIF2B without reducing ε/Gcd6 abundance in the native eIF2B-eIF2 holocomplex. Additionally, β/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2α (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, β/Gcd7 can overcome the toxicity of eIF2(αP) and rescue native eIF2B function when overexpressed with δ/Gcd2 or γ/Gcd1. In aggregate, these findings provide compelling evidence that β/Gcd7 is crucial for binding of substrate by eIF2B in vivo, beyond its dispensable regulatory role in the inhibition of eIF2B by eIF (αP).

Snf1 is the ortholog of mammalian AMP-activated kinase and is responsible for activation of glucose-repressed genes at low glucose levels in budding yeast. We show that Snf1 promotes the formation of phosphorylated alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha-P), a regulator of general and gene-specific translation, by stimulating the function of eIF2alpha kinase Gcn2 during histidine starvation of glucose-grown cells. Thus, eliminating Snf1 or mutating its activation loop lowers Gcn2 kinase activity, reducing the autophosphorylation of Thr-882 in the Gcn2 activation loop, and decreases eIF2alpha-P levels in starved cells. Consistently, eliminating Reg1, a negative regulator of Snf1, provokes Snf1-dependent hyperphosphorylation of both Thr-882 and eIF2alpha. Interestingly, Snf1 also promotes eIF2alpha phosphorylation in the nonpreferred carbon source galactose, but this occurs by inhibition of protein phosphatase 1alpha (PP1alpha; Glc7) and the PP2A-like enzyme Sit4, rather than activation of Gcn2. Both Glc7 and Sit4 physically interact with eIF2alpha in cell extracts, supporting their direct roles as eIF2alpha phosphatases. Our results show that Snf1 modulates the level of eIF2alpha phosphorylation by different mechanisms, depending on the kind of nutrient deprivation existing in cells.

Cyclin-dependent kinase BUR1/BUR2 appears to be the yeast ortholog of P-TEFb, which phosphorylates Ser2 of the RNA Pol II CTD, but the importance of BUR1/BUR2 in CTD phosphorylation is unclear. We show that BUR1/BUR2 is co-transcriptionally recruited to the 5′ end of ARG1 in a manner stimulated by interaction of the BUR1 C-terminus with CTD repeats phosphorylated on Ser5 by KIN28. Impairing BUR1/BUR2 function, or removing the CTD-interaction domain in BUR1, reduces Ser2 phosphorylation in bulk Pol II and eliminates the residual Ser2P in cells lacking the major Ser2 CTD kinase, CTK1. Impairing BUR1/BUR2 or CTK1 evokes a similar reduction of Ser2P in Pol II phosphorylated on Ser5, and in elongating Pol II near the ARG1 promoter. By contrast, CTK1 is responsible for the bulk of Ser2P in total Pol II and at promoter-distal sites. In addition to phosphorylating Ser2 near promoters, BUR1/BUR2 also stimulates Ser2P formation by CTK1 during transcription elongation.

Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix alphaC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2alpha phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of alphaC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of alphaC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.

The late endosome (MVB) plays a key role in coordinating vesicular transport of proteins between the Golgi complex, vacuole/lysosome, and plasma membrane. We found that deleting multiple genes involved in vesicle fusion at the MVB (class C/D vps mutations) impairs transcriptional activation by Gcn4, a global regulator of amino acid biosynthetic genes, by decreasing the ability of chromatin-bound Gcn4 to stimulate preinitiation complex assembly at the promoter. The functions of hybrid activators with Gal4 or VP16 activation domains are diminished in class D mutants as well, suggesting a broader defect in activation. Class E vps mutations, which impair protein sorting at the MVB, also decrease activation by Gcn4, provided they elicit rapid proteolysis of MVB cargo proteins in the aberrant late endosome. By contrast, specifically impairing endocytic trafficking from the plasma membrane, or vesicular transport to the vacuole, has a smaller effect on Gcn4 function. Thus, it appears that decreasing cargo proteins in the MVB through impaired delivery or enhanced degradation, and not merely the failure to transport cargo properly to the vacuole or downregulate plasma membrane proteins by endocytosis, is required to attenuate substantially transcriptional activation by Gcn4.

Summary We report that coactivator SAGA, containing the HAT Gcn5p, occupies the GAL1 and ARG1 coding sequences during transcriptional induction, dependent on PIC assembly and Ser5 phosphorylation of the Pol II CTD. Induction of GAL1 increases H3 acetylation per nucleosome in the ORF, dependent on SAGA integrity but not the alternative Gcn5p-HAT complex ADA. Unexpectedly, H3 acetylation in ARG1 coding sequences does not increase during induction due to the opposing activities of multiple HDAs associated with the ORF. Remarkably, inactivation of Gcn5p decreases nucleosome eviction from both GAL1 and a long (∼8 kb) ORF transcribed from the GAL1 promoter. This is associated with reduced Pol II occupancy at the 3′ end and decreased mRNA production, selectively, for the long ORF. Gcn5p also enhances H3-K4 trimethylation in the ARG1 ORF and bulk histones. Thus, Gcn5p, most likely in SAGA, stimulates modification and eviction of nucleosomes in transcribed coding sequences and promotes Pol II elongation.

The Cyc8p/Tup1p complex mediates repression of diverse genes in Saccharomyces cerevisiae and is recruited by DNA binding proteins specific for the different sets of repressed genes. By screening the yeast deletion library, we identified Cyc8p as a coactivator for Gcn4p, a transcriptional activator of amino acid biosynthetic genes. Deletion of CYC8 confers sensitivity to an inhibitor of isoleucine/valine biosynthesis and impairs activation of Gcn4p-dependent reporters and authentic amino acid biosynthetic target genes. Deletion of TUP1 produces similar but less severe activation defects in vivo. Although expression of Gcn4p is unaffected by deletion of CYC8, chromatin immunoprecipitation assays reveal a strong defect in binding of Gcn4p at the target genes ARG1 and ARG4 in cyc8Delta cells and to a lesser extent in tup1Delta cells. The defects in Gcn4p binding and transcriptional activation in cyc8Delta cells cannot be overcome by Gcn4p overexpression but are partially suppressed in tup1Delta cells. The impairment of Gcn4p binding in cyc8Delta and tup1Delta cells is severe enough to reduce recruitment of SAGA, Srb mediator, TATA binding protein, and RNA polymerase II to the ARG1 and ARG4 promoters, accounting for impaired transcriptional activation of these genes in both mutants. Cyc8p and Tup1p are recruited to the ARG1 and ARG4 promoters, consistent with a direct role for this complex in stimulating Gcn4p occupancy of the upstream activation sequence (UAS). Interestingly, Gcn4p also stimulates binding of Cyc8p/Tup1p at the 3' ends of these genes, raising the possibility that Cyc8p/Tup1p influences transcription elongation. Our findings reveal a novel coactivator function for Cyc8p/Tup1p at the level of activator binding and suggest that Gcn4p may enhance its own binding to the UAS by recruiting Cyc8p/Tup1p.

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. The autoinhibited form of GCN2 is activated in cells starved of amino acids by binding of uncharged tRNA to a histidyl-tRNA synthetase-like domain. Replacement of Arg-794 with Gly in the PK domain (R794G) activates GCN2 independently of tRNA binding. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP. These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving Arg794. This conformational change is associated with intradomain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation.

Transcription of the arginine biosynthetic gene ARG1 is repressed by the ArgR/Mcm1p complex in arginine-replete cells and activated by Gcn4p, a transcription factor induced by starvation for any amino acid. We show that all four subunits of the arginine repressor are recruited to ARG1 by Gcn4p in cells replete with arginine but starved for isoleucine/valine. None of these proteins is recruited to the Gcn4p target genes ARG4 and SNZ1 , which are not regulated by ArgR/Mcm1p. Mcm1p and Arg80p were found in a soluble complex lacking Arg81p and Arg82p, and both Mcm1p and Arg80p were efficiently recruited to ARG1 in wild-type cells in the presence or absence of exogenous arginine, and also in arg81 Δ cells. By contrast, the recruitment of Arg81p and Arg82p was stimulated by exogenous arginine. These findings suggest that Gcn4p constitutively recruits an Mcm1p/Arg80p heterodimer and that efficient assembly of a functional repressor also containing Arg81p and Arg82p occurs only in arginine excess. By recruiting an arginine-regulated repressor, Gcn4p can precisely modulate its activation function at ARG1 according to the availability of arginine.

Gcn4 is a master transcriptional regulator of amino acid and vitamin biosynthetic enzymes subject to the general amino acid control (GAAC), whose expression is upregulated in response to amino acid starvation in Saccharomyces cerevisiae. We found that accumulation of the threonine pathway intermediate β-aspartate semialdehyde (ASA), substrate of homoserine dehydrogenase (Hom6), attenuates the GAAC transcriptional response by accelerating degradation of Gcn4, already an exceedingly unstable protein, in cells starved for isoleucine and valine. The reduction in Gcn4 abundance on ASA accumulation requires Cdk8/Srb10 and Pho85, cyclin-dependent kinases (CDKs) known to mediate rapid turnover of Gcn4 by the proteasome via phosphorylation of the Gcn4 activation domain under nonstarvation conditions. Interestingly, rescue of Gcn4 abundance in hom6 cells by elimination of SRB10 is not accompanied by recovery of transcriptional activation, while equivalent rescue of UAS-bound Gcn4 in hom6 pho85 cells restores greater than wild-type activation of Gcn4 target genes. These and other findings suggest that the two CDKs target different populations of Gcn4 on ASA accumulation, with Srb10 clearing mostly inactive Gcn4 molecules at the promoter that are enriched for sumoylation of the activation domain, and Pho85 clearing molecules unbound to the UAS that include both fully functional and inactive Gcn4 species., Author Summary Transcriptional activator Gcn4 maintains amino acid homeostasis in budding yeast by inducing multiple amino acid biosynthetic pathways in response to starvation for any amino acid—the general amino acid control. Gcn4 abundance is tightly regulated by the interplay between an intricate translational control mechanism, which induces Gcn4 synthesis in starved cells, and a pathway of phosphorylation and ubiquitylation that mediates its rapid degradation by the proteasome. Here, we discovered that accumulation of a threonine biosynthetic pathway intermediate, β-aspartate semialdehyde (ASA), in hom6Δ mutant cells impairs general amino acid control in cells starved for isoleucine and valine by accelerating the already rapid degradation of Gcn4, in a manner requiring its phosphorylation by cyclin-dependent kinases Cdk8/Srb10 and Pho85. Interestingly, our results unveil a division of labor between these two kinases wherein Srb10 primarily targets inactive Gcn4 molecules—presumably damaged under conditions of ASA excess—while Pho85 clears a greater proportion of functional Gcn4 species from the cell. The ability of ASA to inhibit transcriptional induction of threonine pathway enzymes by Gcn4, dampening ASA accumulation and its toxic effects on cell physiology, should be adaptive in the wild when yeast encounters natural antibiotics that target Hom6 enzymatic activity.

GCN2 stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating translation initiation factor 2. GCN2 is activated by binding of uncharged tRNA to a domain related to histidyl-tRNA synthetase (HisRS). The HisRS-like region contains two dimerization domains (HisRS-N and HisRS-C) required for GCN2 function in vivo but dispensable for dimerization by full-length GCN2. Residues corresponding to amino acids at the dimer interface of Escherichia coli HisRS were required for dimerization of recombinant HisRS-N and for tRNA binding by full-length GCN2, suggesting that HisRS-N dimerization promotes tRNA binding and kinase activation. HisRS-N also interacted with the protein kinase (PK) domain, and a deletion impairing this interaction destroyed GCN2 function without reducing tRNA binding; thus, HisRS-N-PK interaction appears to stimulate PK function. The C-terminal domain of GCN2 (C-term) interacted with the PK domain in a manner disrupted by an activating PK mutation (E803V). These results suggest that the C-term is an autoinhibitory domain, counteracted by tRNA binding. We conclude that multiple domain interactions, positive and negative, mediate the activation of GCN2 by uncharged tRNA.

In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.

There is increasing evidence that certain Vacuolar protein sorting (Vps) proteins, factors that mediate vesicular protein trafficking, have additional roles in regulating transcription factors at the endosome. We found that yeast mutants lacking the phosphatidylinositol 3-phosphate [PI(3)P] kinase Vps34 or its associated protein kinase Vps15 display multiple phenotypes indicating impaired transcription elongation. These phenotypes include reduced mRNA production from long or G+C-rich coding sequences (CDS) without affecting the associated GAL1 promoter activity, and a reduced rate of RNA polymerase II (Pol II) progression through lacZ CDS in vivo. Consistent with reported genetic interactions with mutations affecting the histone acetyltransferase complex NuA4, vps15Δ and vps34Δ mutations reduce NuA4 occupancy in certain transcribed CDS. vps15Δ and vps34Δ mutants also exhibit impaired localization of the induced GAL1 gene to the nuclear periphery. We found unexpectedly that, similar to known transcription elongation factors, these and several other Vps factors can be cross-linked to the CDS of genes induced by Gcn4 or Gal4 in a manner dependent on transcriptional induction and stimulated by Cdk7/Kin28-dependent phosphorylation of the Pol II C-terminal domain (CTD). We also observed colocalization of a fraction of Vps15-GFP and Vps34-GFP with nuclear pores at nucleus–vacuole (NV) junctions in live cells. These findings suggest that Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed chromatin.

Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment.

The RAD25 gene of Saccharomyces cerevisiae is required for excision repair of ultraviolet-damaged DNA and, in addition, is essential for viability. RAD25 shares a high degree of homology with the human ERCC3/XPBC-encoded protein, and the yeast and human proteins resemble one another in containing the conserved ATPase/DNA helicase sequence motifs. To determine the nature of the essential role of RAD25, we have isolated a recessive temperature-sensitive conditional lethal mutation of the gene and have examined its effect on transcription. Upon shift to the nonpermissive temperature, the rad25 temperature-sensitive (ts) mutant stops growth rapidly and shows a large decrease in the synthesis of poly(A)+ RNA. Transcription of a large number of yeast genes, including HIS3, TRP3, STE2, MET19, RAD23, CDC9, and ACT1 is inhibited at the restrictive temperature in the rad25 ts mutant, and the galactose-inducible synthesis of GAL7 and GAL10 mRNAs is also severely affected by the loss of RAD25 activity. These findings implicate a general requirement of RAD25 in RNA polymerase II transcription.