Cryo-EM structures of the plant plastid-encoded RNA polymerase.

Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.

Structural basis of exoribonuclease-mediated mRNA transcription termination.

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

Structural basis for intrinsic transcription termination.

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.

An SI3-σ arch stabilizes cyanobacteria transcription initiation complex.

Multisubunit RNA polymerases (RNAPs) associate with initiation factors (σ in bacteria) to start transcription. The σ factors are responsible for recognizing and unwinding promoter DNA in all bacterial RNAPs. Here, we report two cryo-EM structures of cyanobacterial transcription initiation complexes at near-atomic resolutions. The structures show that cyanobacterial RNAP forms an "SI3-σ" arch interaction between domain 2 of σA (σ2) and sequence insertion 3 (SI3) in the mobile catalytic domain Trigger Loop (TL). The "SI3-σ" arch facilitates transcription initiation from promoters of different classes through sealing the main cleft and thereby stabilizing the RNAP-promoter DNA open complex. Disruption of the "SI3-σ" arch disturbs cyanobacteria growth and stress response. Our study reports the structure of cyanobacterial RNAP and a unique mechanism for its transcription initiation. Our data suggest functional plasticity of SI3 and provide the foundation for further research into cyanobacterial and chloroplast transcription.

The structural mechanism for transcription activation by Caulobacter crescentus GcrA.

Canonical bacterial transcription activators bind to their cognate cis elements at the upstream of transcription start site (TSS) in a form of dimer. Caulobacter crescentus GcrA, a non-canonical transcription activator, can activate transcription from promoters harboring its cis element at the upstream or downstream of TSS in a form of monomer. We determined two cryo-EM structures of C. crescentus GcrA-bound transcription activation complexes, GcrA TACU and GcrA TACD, which comprise GcrA, RNAP, σ70 and promoter DNA with GcrA cis elements at either the upstream or downstream of TSS at 3.6 and 3.8 Å, respectively. In the GcrA-TACU structure, GcrA makes bipartite interactions with both σ70 domain 2 (σ702) and its cis element, while in the GcrA-TACD structure, GcrA retains interaction with σ702 but loses the interaction with its cis element. Our results suggest that GcrA likely forms a functionally specialized GcrA-RNAP-σA holoenzyme, in which GcrA first locates its cis element and then facilitates RNAP to load on core promoter at its proximal region. The sequence-specific interaction of GcrA and DNA is disrupted either at the stage of RPo formation or promoter escape depending on the location of GcrA cis elements relative to TSS.

A Noncoding A-to-U Kozak Site Change Related to the High Transmissibility of Alpha, Delta, and Omicron VOCs.

Three prevalent SARS-CoV-2 variants of concern (VOCs) emerged and caused epidemic waves. It is essential to uncover advantageous mutations that cause the high transmissibility of VOCs. However, viral mutations are tightly linked, so traditional population genetic methods, including machine learning-based methods, cannot reliably detect mutations conferring a fitness advantage. In this study, we developed an approach based on the sequential occurrence order of mutations and the accelerated furcation rate in the pandemic-scale phylogenomic tree. We analyzed 3,777,753 high-quality SARS-CoV-2 genomic sequences and the epidemiology metadata using the Coronavirus GenBrowser. We found that two noncoding mutations at the same position (g.a28271-/u) may be crucial to the high transmissibility of Alpha, Delta, and Omicron VOCs although the noncoding mutations alone cannot increase viral transmissibility. Both mutations cause an A-to-U change at the core position -3 of the Kozak sequence of the N gene and significantly reduce the protein expression ratio of ORF9b to N. Using a convergent evolutionary analysis, we found that g.a28271-/u, S:p.P681H/R, and N:p.R203K/M occur independently on three VOC lineages, suggesting that coordinated changes of S, N, and ORF9b proteins are crucial to high viral transmissibility. Our results provide new insights into high viral transmissibility co-modulated by advantageous noncoding and nonsynonymous changes.

Comparative proteomic analysis of two divergent strains provides insights into thermotolerance mechanisms of Ganoderma lingzhi.

Heat stress (HS) is a major abiotic factor influencing fungal growth and metabolism. However, the genetic basis of thermotolerance in Ganoderma lingzhi (G. lingzhi) remains largely unknown. In this study, we investigated the thermotolerance capacities of 21 G. lingzhi strains and screened the thermo-tolerant (S566) and heat-sensitive (Z381) strains. The mycelia of S566 and Z381 were collected and subjected to a tandem mass tag (TMT)-based proteome assay. We identified 1493 differentially expressed proteins (DEPs), with 376 and 395 DEPs specific to the heat-tolerant and heat-susceptible genotypes, respectively. In the heat-tolerant genotype, upregulated proteins were linked to stimulus regulation and response. Proteins related to oxidative phosphorylation, glycosylphosphatidylinositol-anchor biosynthesis, and cell wall macromolecule metabolism were downregulated in susceptible genotypes. After HS, the mycelial growth of the heat-sensitive Z381 strain was inhibited, and mitochondrial cristae and cell wall integrity of this strain were severely impaired, suggesting that HS may inhibit mycelial growth of Z381 by damaging the cell wall and mitochondrial structure. Furthermore, thermotolerance-related regulatory pathways were explored by analyzing the protein-protein interaction network of DEPs considered to participate in the controlling the thermotolerance capacity. This study provides insights into G. lingzhi thermotolerance mechanisms and a basis for breeding a thermotolerant germplasm bank for G. lingzhi and other fungi.

CueR activates transcription through a DNA distortion mechanism.

The MerR-family transcription factors (TFs) are a large group of bacterial proteins responding to cellular metal ions and multiple antibiotics by binding within central RNA polymerase-binding regions of a promoter. While most TFs alter transcription through protein-protein interactions, MerR TFs are capable of reshaping promoter DNA. To address the question of which mechanism prevails, we determined two cryo-EM structures of transcription activation complexes (TAC) comprising Escherichia coli CueR (a prototype MerR TF), RNAP holoenzyme and promoter DNA. The structures reveal that this TF promotes productive promoter-polymerase association without canonical protein-protein contacts seen between other activator proteins and RNAP. Instead, CueR realigns the key promoter elements in the transcription activation complex by clamp-like protein-DNA interactions: these induce four distinct kinks that ultimately position the -10 element for formation of the transcription bubble. These structural and biochemical results provide strong support for the DNA distortion paradigm of allosteric transcriptional control by MerR TFs.

Study on the inhibitory effect of fermentation extract of Microporus vernicipes on Candida albicans.

Candida albicans is one of the most common species of Candida, which cause various mucosal and systemic infectious diseases. However, the resistance rate to existing clinical antifungal drugs gradually increases in C. albicans. Therefore, new antifungal drugs must be developed to solve the current problem. This study discovered that the solid fermented ethyl acetate crude extract of Microporus vernicipes had inhibitory activity on C. albicans. This study determined that the Mv5 components had significantly inhibited the activity of C. albicans using column chromatography separation component screening. The components included 23 compounds of fatty acids and their derivatives, alkaloids, phenols, and other classes using ultra-high performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HR-MS) analysis, with fatty acids constituting the primary components. The mechanism of action showed that the minimum inhibitory concentration (MIC) of Mv5 components against C. albicans was 15.63 µg/mL, while minimum fungicidal concentration (MFC) was 31.25 µg/mL. Mv5 components can inhibit the early biofilm formation and destroy the mature biofilm structure. It can inhibit the germ tube growth of C. albicans, thereby inhibiting the transformation of yeast morphology to hyphae. We detected 193 differentially expressed genes, including 156 upregulated and 37 downregulated genes in the Mv5 components of the MIC concentration group. We detected 391 differentially expressed genes, including 334 upregulated and 57 downregulated expression genes in the MFC concentration group. Among these differentially expressed genes, the genes related to mycelium and biofilm formation were significantly downregulated. GO enrichment analysis presented that single-organism process metabolic process, and cellular processes were the biological processes with the most gene enrichment. Kyoto Encyclopedia of Genes and Genomes (KEGG)of Mv5 components were mainly enriched in metabolic pathways, such as meiosis yeast and amino acid metabolism. Therefore, it is believed that the fermentation extract of M. vernicipes inhibits C. albicans, which can provide clues for developing effective antifungal drugs.

Crystal structures and biochemical analyses of the bacterial arginine dihydrolase ArgZ suggests a "bond rotation" catalytic mechanism.

A recently discovered ornithine-ammonia cycle (OAC) serves as a conduit in the nitrogen storage and remobilization machinery in cyanobacteria. The OAC involves an arginine catabolic reaction catalyzed by the arginine dihydrolase ArgZ whose catalytic mechanism is unknown. Here we determined the crystal structures at 1.2-3.0 Å of unliganded ArgZ from the cyanobacterium Synechocystis sp. PCC6803 and of ArgZ complexed with its substrate arginine, a covalently linked reaction intermediate, or the reaction product ornithine. The structures reveal that a key residue, Asn71, in the ArgZ active center functions as the determinant distinguishing ArgZ from other members of the guanidino group-modifying enzyme superfamily. The structures, along with biochemical evidence from enzymatic assays coupled with electrospray ionization MS techniques, further suggest that ArgZ-catalyzed conversion of arginine to ornithine, ammonia, and carbon dioxide consists of two successive cycles of amine hydrolysis. Finally, we show that arginine dihydrolases are broadly distributed among bacteria and metazoans, suggesting that the OAC may be frequently used for redistribution of nitrogen from arginine catabolism or nitrogen fixation.

Structural insights into the unique mechanism of transcription activation by Caulobacter crescentus GcrA.

Canonical bacterial transcription activators bind to non-transcribed promoter elements to increase transcription of their target genes. Here we report crystal structures of binary complexes comprising domains of Caulobacter crescentus GcrA, a noncanonical bacterial transcription factor, that support a novel mechanism for transcription activation through the preferential binding of methylated cis-regulatory elements and the promotion of open complex formation through an interaction with region 2 of the principal σ factor, σ70. We present crystal structures of the C-terminal, σ factor-interacting domain (GcrA-SID) in complex with domain 2 of σ70 (σ702), and the N-terminal, DNA-binding domain (GcrA-DBD) in complex with methylated double-stranded DNA (dsDNA). The structures reveal interactions essential for transcription activation and DNA recognition by GcrA. These structures, along with mutational analyses, support a mechanism of transcription activation in which GcrA associates with RNA polymerase (RNAP) prior to promoter binding through GcrA-SID, arming RNAP with a flexible GcrA-DBD. The RNAP-GcrA complex then binds and activates target promoters harboring a methylated GcrA binding site either upstream or downstream of the transcription start site.

Effect of Ganoderma Lucidum Preparation on the Behavior,Biochemistry,and Autoimmune Parameters of Mouse Models of APP/PS1 Double Transgenic Alzheimer's Disease.

Objective To evaluate the efficacy of Ganoderma lucidum preparation on the behaviors,biochemistry,and autoimmunity parameters of mouse models of APP/PS-1 double transgenic Alzheimer's disease(AD).Methods A total of 44 4-month-old APP/PS-1 double transgenic AD mice were randomly divided into AD model group,Aricept group,Ganoderma lucidum middle-dose(LZ-M)group,and Ganoderma lucidum high-dose(LZ-H)group,with 11 mice in each group.In addition,10 4-month-old C57BL/6 mice were used as the control group.Water maze test was conducted to observe the behavior changes,and the protein expressions in brain tissues were detected by Western blot analysis.The autoimmune indicators were detected by indirect immunofluorescence method.Results In the navigation experiment,the time of finding the platform was gradually shortened since the 2nd day in the control,LZ-H,and LZ-M groups,and the time of searching the platform in the AD model group gradually increased.On the 5th day,the time of finding platform was significantly shorter in control group (t=5.607,P=0.000) and LZ-H group(t=2.750,P=0.010)than AD model group.In the space exploration experiment,the number of crossing the target platform(t=2.452,P=0.025)and the residence time in the target quadrant(t=2.530,P=0.020)in AD model group mice was significantly smaller/shorter than those in control group;in addition,the number of crossing the target platform in the AD model group was significantly smaller than that in LZ-H group(t=2.317,P=0.030)and LZ-M group(t=2.443,P=0.030),while the residence time in target quadrant decreased significantly(t=2.770,P=0.020)compared with LZ-H group;the number of crossing through the target platform quadrant(t=2.493,P=0.022)and residence time in the target quadrant(t=2.683,P=0.015)in LZ-H group were significantly higher than in Aricept group.Western blot analysis showed that the expression of ApoA1 in the brain tissues of mice in LZ-H and LZ-M groups were significantly higher than those in AD model group(P<0.01,P<0.05);Aß-40 expression in LZ-H group was significantly lower than that in AD model group(P<0.05);the expressions of Syt1,ApoE,and ABCA1 in brain tissues of mice in LZ-H group were significantly higher than those in model group(P<0.01,P<0.05).The plasma IgG level in Aricept group(t=30.945,P=0.000),LZ-M group(t=25.639,P=0.000)and LZ-H group(t=4.689,P=0.001)were significantly higher than that in the control group.Conclusion Ganoderma lucidum preparation can improve behavior disorders of AD model mice,promote the expressions of ApoA1,ApoE and Syt1,inhibit the expression of Aß-40 protein,and improve the autoimmune function.

Pathological Changes in APP/PS-1 Transgenic Mouse Models of Alzheimer's Disease Treated with Ganoderma Lucidum Preparation.

Objective To explore the efficacy of ganoderma lucidum preparation(Ling Zhi) in treating APP/PS-1 transgenic mouse models of Alzheimer's disease(AD).Methods APP/PS-1 transgenic mice of 4 months were randomly divided into model group,ganoderma lucidum treatment groups,including high [2250 mg/(kg·d)] and middle [750 mg/(kg·d)] dose groups,i.e.LZ-H and LZ-M groups,and the positive control group(treated with donepezil hydrochloride [2 mg/(kg·d)]).In addition,C57BL/6J wild mice were selected as normal group.The animals were administered for 4 months.Histopathological examinations including hematoxylin-eosin(HE) staining,immunohistochemistry,special staining,and electron microscopy were applied,and then the pathological morphology and structures in different groups were compared. Results The senile plaques and neurofibrillar tangles in the cerebrum and cerebellum were dissolved or disappeared in LZ-H and LZ-M groups.Decrease of amyloid angiopathy was found in LZ-H and LZ-M groups.The immature neurons appeared more in hippocampus and dentate nucleus of LZ-H and LZ-M groups than those in AD model and donepezil hydrochloride groups(hippcampus:F=1.738,P=0.016;dentate nucleus:F=1.924,P=0.026),and these immature neurons differentiated to be neurons.More Purkinje cells loss occurred in AD model mice than that in LZ-H and LZ-M groups(F=9.46,P=0.007;F=9.46,P=0.010).The LZ-H and LZ-M groups had more new neuron stem cells grown up in cerebellum.Electromicroscopic examination showed the hippocampal neurons in LZ-H and LZ-M group were integrated,the nuclear membrane was intact,and the mitochondria in the cytoplasm,endoplasmic reticulum,Golgi bodies,microtubules,and synapses were also complete.The microglial cell showed no abnormality.No toxicity appeared in the pathological specimens of mice treated with ganoderma lucidum preparation.Conclusion The ganoderma lucidum preparation can dissolve and decline or dismiss the senile plaques and neurofibrillar tangles in the brain of AD mice and also reduce the amyloid angiopathy.

One-Step Synthesis of Hydrogel Adhesive with Acid-Responsive Tannin Release for Diabetic Oral Mucosa Defects Healing.

The complex preparation, weak wet tissue adhesion, and limited biological activity of traditional oral wound dressings usually impede their efficient treatment and healing for diabetic oral mucosal defects. To overcome these problems, a novel hydrogel adhesive (named CFT hydrogel) is rapidly constructed using a one-step method based on dual-dynamic covalent cross-linking. Compared with the commercial oral patches, the CFT hydrogel shows superior in vivo (rat tongue) wet tissue adhesion performance. Additionally, the CFT hydrogel exhibits unique acid-responsive properties, thereby facilitating the release of bioactive molecule tannic acid in the acidic diabetic wound microenvironment. And a series of in vitro experiments substantiate the favorable biocompatibility and bioactivity properties (including antibacterial, antioxidative, anti-inflammatory, and angiogenetic effects) exhibited by CFT hydrogel. Moreover, in vivo experiments conducted on a diabetic rat model with oral mucosal defects demonstrate that the CFT hydrogel exhibits significant efficacy in protecting against mucosal wounds, alleviating inflammatory reactions, thereby facilitating the wound-healing process. Taken together, this study provides a promising and comprehensive therapeutic option with great potential for the clinical management of oral mucosa defects in diabetic patients.

DNA looping mediates cooperative transcription activation.

Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here we report two cryo-electron microscopy structures of Anabaena transcription activation complexes (TACs): NtcA-TAC composed of RNAP holoenzyme, promoter and a global activator NtcA, and NtcA-NtcB-TAC comprising an extra context-specific regulator, NtcB. Structural analysis showed that NtcA binding makes the promoter DNA bend by ∼50°, which facilitates RNAP to contact NtcB at the distal upstream NtcB box. The sequential binding of NtcA and NtcB induces looping back of promoter DNA towards RNAP, enabling the assembly of a fully activated TAC bound with two activators. Together with biochemical assays, we propose a 'DNA looping' mechanism of cooperative transcription activation in bacteria.

Rhabdovirus encoded glycoprotein induces and harnesses host antiviral autophagy for maintaining its compatible infection.

Macroautophagy/autophagy has been recognized as a central antiviral defense mechanism in plant, which involves complex interactions between viral proteins and host factors. Rhabdoviruses are single-stranded RNA viruses, and the infection causes serious harm to public health, livestock, and crop production. However, little is known about the role of autophagy in the defense against rhabdovirus infection by plant. In this work, we showed that Rice stripe mosaic cytorhabdovirus(RSMV) activated autophagy in plants and that autophagy served as an indispensable defense mechanism during RSMV infection. We identified RSMV glycoprotein as an autophagy inducer that interacted with OsSnRK1B and promoted the kinase activity of OsSnRK1B on OsATG6b. RSMV glycoprotein was toxic to rice cells and its targeted degradation by OsATG6b-mediated autophagy was essential to restrict the viral titer in plants. Importantly, SnRK1-glycoprotein and ATG6-glycoprotein interactions were well-conserved between several other rhabdoviruses and plants. Together, our data support a model that SnRK1 senses rhabdovirus glycoprotein for autophagy initiation, while ATG6 mediates targeted degradation of viral glycoprotein. This conserved mechanism ensures compatible infection by limiting the toxicity of viral glycoprotein and restricting the infection of rhabdoviruses.Abbreviations: AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; ANOVA: analysis of variance; ATG: autophagy related; AZD: AZD8055; BiFC: bimolecular fluorescence complementation; BYSMV: barley yellow striate mosaic virus; Co-IP: co-immunoprecipitation; ConA: concanamycin A; CTD: C-terminal domain; DEX: dexamethasone; DMSO: dimethyl sulfoxide; G: glycoprotein; GFP: green fluorescent protein; MD: middle domain; MDC: monodansylcadaverine; NTD: N-terminal domain; OE: over expression; Os: Oryza sativa; PBS: phosphate-buffered saline; PtdIns3K: class III phosphatidylinositol-3-kinase; qRT-PCR: quantitative real-time reverse-transcription PCR; RFP: red fluorescent protein; RSMV: rice stripe mosaic virus; RSV: rice stripe virus; SGS3: suppressor of gene silencing 3; SnRK1: sucrose nonfermenting1-related protein kinase1; SYNV: sonchus yellow net virus; TEM: transmission electron microscopy; TM: transmembrane region; TOR: target of rapamycin; TRV: tobacco rattle virus; TYMaV: tomato yellow mottle-associated virus; VSV: vesicular stomatitis virus; WT: wild type; Y2H: yeast two-hybrid; YFP: yellow fluorescent protein.

Is Lidocaine Patch Beneficial for Postoperative Pain?: A Meta-analysis of Randomized Clinical Trials.

OBJECTIVES: The aim of this meta-analysis was to evaluate whether a lidocaine patch is beneficial for postoperative pain as an option for multimodal analgesia. METHODS: Information was obtained from PubMed, Embase, and the Cochrane Central Register of Controlled Trials for clinical randomized controlled trials of lidocaine patches for postoperative pain (as of March 2022). Two researchers independently completed study screening, risk bias assessment, and data extraction. Review Manager (version 5.4, Cochrane Collaboration) was used to conduct the meta-analysis. The evaluation metrics were postoperative pain scores, opioid consumption, and patient satisfaction. RESULTS: Sixteen randomized controlled trials were included, and data from 918 patients were available. Pain scores differed between the 2 groups at 12, 24, and 48 hours postoperatively, and the pain scores of the lidocaine patch group were significantly lower (mean difference [MD]=-1.32 [95% CI, -1.96 to -0.68], P <0.0001; I2 =92%) at 12 hours after the operation; (MD=-1.23 [95% CI, -1.72 to -0.75], P <0.00001; I2 =92%) at 24 hours after the operation; and (MD=-0.25 [95% CI,-0.29 to -0.21], P <0.00001; I2 =98%) at 48 hours after the operation. In addition, the lidocaine patch group had decreased opioid requirements (MD=-3.57 [95% CI, -5.06 to -2.09], P <0.00001; I2 =96%). The lidocaine patch group seemed to be more satisfied, but there was no statistically significant difference (risk ratio, 1.50 [95% CI, 0.74 to 3.05], P =0.26) between the groups. DISCUSSION: Lidocaine patches are beneficial for postoperative pain and can be used in multimodal analgesia to reduce opioid use, but there is no significant increase in patient satisfaction with pain control. More data are needed to support this conclusion due to the large heterogeneity in the present study.

A cryo-EM structure of KTF1-bound polymerase V transcription elongation complex.

De novo DNA methylation in plants relies on transcription of RNA polymerase V (Pol V) along with KTF1, which produce long non-coding RNAs for recruitment and assembly of the DNA methylation machinery. Here, we report a cryo-EM structure of the Pol V transcription elongation complex bound to KTF1. The structure reveals the conformation of the structural motifs in the active site of Pol V that accounts for its inferior RNA-extension ability. The structure also reveals structural features of Pol V that prevent it from interacting with the transcription factors of Pol II and Pol IV. The KOW5 domain of KTF1 binds near the RNA exit channel of Pol V providing a scaffold for the proposed recruitment of Argonaute proteins to initiate the assembly of the DNA methylation machinery. The structure provides insight into the Pol V transcription elongation process and the role of KTF1 during Pol V transcription-coupled DNA methylation.

Development of tannin-bridged cerium oxide microcubes-chitosan cryogel as a multifunctional wound dressing.

Efficient resolution of oxidative stress, inflammation, and bacterial infections is crucial for wound healing. To surmount these problems, tannic acid (TA)-bridged CeO2 microcubes and chitosan (CS) (CS-TA@CeO2) cryogel was fabricated through hydrogen bonding interactions as a multifunctional wound dressing. Successful introduction and uniform incorporation TA@CeO2 microtubules enter the CS network. Thus-obtained CS-TA@CeO2 cryogels displayed a suitable porous structure and swelling rate. Cryogels has excellent tissue adhesion, blood cell coagulation and hemostasis, anti-infection, and cell recruitment functions. In addition, the cryogel also showed good antibacterial activity against gram-positive bacteria and gram-negative bacteria. Based on the in vivo study of the multifunctional mixed cryogels, it promotes fibroblasts' adhesion and proliferation and significantly improves cell proliferation and tissue remodelling in wound beds. Furthermore, the chronic wound healing process in infected full-thickness skin defect models showed that cryogels significantly enhanced angiogenesis, collagen deposition and granulation tissue formation by providing a large amount of antioxidant activity. Therefore, this multifunctional mixed cryogels has potential clinical application value.

Pseudomonas aeruginosa SutA wedges RNAP lobe domain open to facilitate promoter DNA unwinding.

Pseudomonas aeruginosa (Pae) SutA adapts bacteria to hypoxia and nutrition-limited environment during chronic infection by increasing transcription activity of an RNA polymerase (RNAP) holoenzyme comprising the stress-responsive σ factor σS (RNAP-σS). SutA shows no homology to previously characterized RNAP-binding proteins. The structure and mode of action of SutA remain unclear. Here we determined cryo-EM structures of Pae RNAP-σS holoenzyme, Pae RNAP-σS holoenzyme complexed with SutA, and Pae RNAP-σS transcription initiation complex comprising SutA. The structures show SutA pinches RNAP-ß protrusion and facilitates promoter unwinding by wedging RNAP-ß lobe open. Our results demonstrate that SutA clears an energetic barrier to facilitate promoter unwinding of RNAP-σS holoenzyme.