Identification of FtfL as a novel target of berberine in intestinal bacteria.

BACKGROUND: Berberine (BBR) is a commonly used anti-intestinal inflammation drug, and its anti-cancer activity has been found recently. BBR can intervene and control malignant colorectal cancer (CRC) through intestinal microbes, but the direct molecular target and related mechanism are unclear. This study aimed to identify the target of BBR and dissect related mechanisms against the occurrence and development of CRC from the perspective of intestinal microorganisms. RESULTS: Here, we found that BBR inhibits the growth of several CRC-driving bacteria, especially Peptostreptococcus anaerobius. By using a biotin-conjugated BBR derivative, we identified the protein FtfL (formate tetrahydrofolate ligase), a key enzyme in C1 metabolism, is the molecular target of BBR in P. anaerobius. BBR exhibits strong binding affinity and potent inhibition on FtfL. Based on this, we determined the crystal structure of PaFtfL (P. anaerobius FtfL)-BBR complex and found that BBR can not only interfere with the conformational flexibility of PaFtfL tetramer by wedging the tetramer interface but also compete with its substrate ATP for binding within the active center. In addition, the enzymatic activities of FtfL homologous proteins in human tumor cells can also be inhibited by BBR. CONCLUSIONS: In summary, our study has identified FtfL as a direct target of BBR and uncovered molecular mechanisms involved in the anti-CRC of BBR. BBR interferes with intestinal pathogenic bacteria by targeting FtfLs, suggesting a new means for controlling the occurrence and development of CRC.

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.

Bacterial MerR family transcription regulators: activationby distortion.

Transcription factors (TFs) modulate gene expression by regulating the accessibility of promoter DNA to RNA polymerases (RNAPs) in bacteria. The MerR family TFs are a large class of bacterial proteins unique in their physiological functions and molecular action: they function as transcription repressors under normal circumstances, but rapidly transform to transcription activators under various cellular triggers, including oxidative stress, imbalance of cellular metal ions, and antibiotic challenge. The promoters regulated by MerR TFs typically contain an abnormal long spacer between the -35 and -10 elements, where MerR TFs bind and regulate transcription activity through unique mechanisms. In this review, we summarize the function, ligand reception, DNA recognition, and molecular mechanism of transcription regulation of MerR-family TFs.

Pol IV and RDR2: A two-RNA-polymerase machine that produces double-stranded RNA.

DNA methylation affects gene expression and maintains genome integrity. The DNA-dependent RNA polymerase IV (Pol IV), together with the RNA-dependent RNA polymerase RDR2, produces double-stranded small interfering RNA precursors essential for establishing and maintaining DNA methylation in plants. We determined the cryo­electron microscopy structures of the Pol IV­RDR2 holoenzyme and the backtracked transcription elongation complex. These structures reveal that Pol IV and RDR2 form a complex with their active sites connected by an interpolymerase channel, through which the Pol IV­generated transcript is handed over to the RDR2 active site after being backtracked, where it is used as the template for double-stranded RNA (dsRNA) synthesis. Our results describe a 'backtracking-triggered RNA channeling' mechanism underlying dsRNA synthesis and also shed light on the evolutionary trajectory of eukaryotic RNA polymerases.

DNA crosslinking and recombination-activating genes 1/2 (RAG1/2) are required for oncogenic splicing in acute lymphoblastic leukemia.

BACKGROUND: Abnormal alternative splicing is frequently associated with carcinogenesis. In B-cell acute lymphoblastic leukemia (B-ALL), double homeobox 4 fused with immunoglobulin heavy chain (DUX4/IGH) can lead to the aberrant production of E-26 transformation-specific family related gene abnormal transcript (ERGalt ) and other splicing variants. However, the molecular mechanism underpinning this process remains elusive. Here, we aimed to know how DUX4/IGH triggers abnormal splicing in leukemia. METHODS: The differential intron retention analysis was conducted to identify novel DUX4/IGH-driven splicing in B-ALL patients. X-ray crystallography, small angle X-ray scattering (SAXS), and analytical ultracentrifugation were used to investigate how DUX4/IGH recognize double DUX4 responsive element (DRE)-DRE sites. The ERGalt biogenesis and B-cell differentiation assays were performed to characterize the DUX4/IGH crosslinking activity. To check whether recombination-activating gene 1/2 (RAG1/2) was required for DUX4/IGH-driven splicing, the proximity ligation assay, co-immunoprecipitation, mammalian two hybrid characterizations, in vitro RAG1/2 cleavage, and shRNA knock-down assays were performed. RESULTS: We reported previously unrecognized intron retention events in C-type lectin domain family 12, member A abnormal transcript (CLEC12Aalt ) and chromosome 6 open reading frame 89 abnormal transcript (C6orf89alt ), where also harbored repetitive DRE-DRE sites. Supportively, X-ray crystallography and SAXS characterization revealed that DUX4 homeobox domain (HD)1-HD2 might dimerize into a dumbbell-shape trans configuration to crosslink two adjacent DRE sites. Impaired DUX4/IGH-mediated crosslinking abolishes ERGalt , CLEC12Aalt , and C6orf89alt biogenesis, resulting in marked alleviation of its inhibitory effect on B-cell differentiation. Furthermore, we also observed a rare RAG1/2-mediated recombination signal sequence-like DNA edition in DUX4/IGH target genes. Supportively, shRNA knock-down of RAG1/2 in leukemic Reh cells consistently impaired the biogenesis of ERGalt , CLEC12Aalt , and C6orf89alt . CONCLUSIONS: All these results suggest that DUX4/IGH-driven DNA crosslinking is required for RAG1/2 recruitment onto the double tandem DRE-DRE sites, catalyzing V(D)J-like recombination and oncogenic splicing in acute lymphoblastic leukemia.

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.

The bacterial multidrug resistance regulator BmrR distorts promoter DNA to activate transcription.

The MerR-family proteins represent a unique family of bacteria transcription factors (TFs), which activate transcription in a manner distinct from canonical ones. Here, we report a cryo-EM structure of a B. subtilis transcription activation complex comprising B. subtilis six-subunit (2αßß'ωε) RNA Polymerase (RNAP) core enzyme, σA, a promoter DNA, and the ligand-bound B. subtilis BmrR, a prototype of MerR-family TFs. The structure reveals that RNAP and BmrR recognize the upstream promoter DNA from opposite faces and induce four significant kinks from the -35 element to the -10 element of the promoter DNA in a cooperative manner, which restores otherwise inactive promoter activity by shortening the length of promoter non-optimal -35/-10 spacer. Our structure supports a DNA-distortion and RNAP-non-contact paradigm of transcriptional activation by MerR TFs.

In Vivo Evolution of CTX-M-215, a Novel Narrow-Spectrum ß-Lactamase in an Escherichia coli Clinical Isolate Conferring Resistance to Mecillinam.

Here, we report a novel narrow-spectrum ß-lactamase CTX-M-215 identified in an Escherichia coli clinical isolate in China and conferring high-level resistance to mecillinam but not to cefotaxime. CTX-M-215 differed from CTX-M-125, a CTX-M extended-spectrum ß-lactamase (ESBL), by an N132D substitution, which decreased hydrolytic activities toward penicillins and cephalosporins except for mecillinam. High similarity was observed between CTX-M-215- and CTX-M-125-bearing plasmids, carried by different isolates in the same patient, indicating in vivo evolution of CTX-M-215 from CTX-M-125.

Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor.

σS is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σS-mediated transcription requires a σS-specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an Escherichia coli transcription activation complex (E. coli Crl-TAC) comprising E. coli σS-RNA polymerase (σS-RNAP) holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with domain 2 of σS (σS2) and the RNAP core enzyme, but does not contact promoter DNA. Results from subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicate that Crl stabilizes key structural motifs within σS2 to promote the assembly of the σS-RNAP holoenzyme and also to facilitate formation of an RNA polymerase-promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.

Tropane alkaloids biosynthesis involves an unusual type III polyketide synthase and non-enzymatic condensation.

The skeleton of tropane alkaloids is derived from ornithine-derived N-methylpyrrolinium and two malonyl-CoA units. The enzymatic mechanism that connects N-methylpyrrolinium and malonyl-CoA units remains unknown. Here, we report the characterization of three pyrrolidine ketide synthases (PYKS), AaPYKS, DsPYKS, and AbPYKS, from three different hyoscyamine- and scopolamine-producing plants. By examining the crystal structure and biochemical activity of AaPYKS, we show that the reaction mechanism involves PYKS-mediated malonyl-CoA condensation to generate a 3-oxo-glutaric acid intermediate that can undergo non-enzymatic Mannich-like condensation with N-methylpyrrolinium to yield the racemic 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This study therefore provides a long sought-after biosynthetic mechanism to explain condensation between N-methylpyrrolinium and acetate units and, more importantly, identifies an unusual plant type III polyketide synthase that can only catalyze one round of malonyl-CoA condensation.

Structural basis for transcription antitermination at bacterial intrinsic terminator.

Bacteriophages typically hijack the host bacterial transcriptional machinery to regulate their own gene expression and that of the host bacteria. The structural basis for bacteriophage protein-mediated transcription regulation-in particular transcription antitermination-is largely unknown. Here we report the 3.4 Å and 4.0 Å cryo-EM structures of two bacterial transcription elongation complexes (P7-NusA-TEC and P7-TEC) comprising the bacteriophage protein P7, a master host-transcription regulator encoded by bacteriophage Xp10 of the rice pathogen Xanthomonas oryzae pv. Oryzae (Xoo) and discuss the mechanisms by which P7 modulates the host bacterial RNAP. The structures together with biochemical evidence demonstrate that P7 prevents transcription termination by plugging up the RNAP RNA-exit channel and impeding RNA-hairpin formation at the intrinsic terminator. Moreover, P7 inhibits transcription initiation by restraining RNAP-clamp motions. Our study reveals the structural basis for transcription antitermination by phage proteins and provides insights into bacterial transcription regulation.

Structures and mechanism of transcription initiation by bacterial ECF factors.

Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE-the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.

Structural basis for transcription initiation by bacterial ECF σ factors.

Bacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated transcription initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis (Mtb) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σH (σH-RNAP) at 2.7 Å, and solve another crystal structure of a transcription initiation complex of Mtb σH-RNAP (σH-RPo) comprising promoter DNA and an RNA primer at 2.8 Å. The two structures together reveal the interactions between σH and RNAP that are essential for σH-RNAP holoenzyme assembly as well as the interactions between σH-RNAP and promoter DNA responsible for stringent promoter recognition and for promoter unwinding. Our study establishes that ECF σ factors and primary σ factors employ distinct mechanisms for promoter recognition and for promoter unwinding.

Epicatechin isolated from Tripterygium wilfordii extract reduces tau-GFP-induced neurotoxicity in zebrafish embryo through the activation of Nrf2.

Tau plays important roles in the assembly and stabilization of the microtubule structure to facilitate axonal transport in mammalian brain. The intracellular tau aggregates to form paired helical filaments leading to neurodegenerative disorders, collectively called tauopathies. In our previous report, we established a zebrafish model to express tau-GFP to induce neuronal death, which could be directly traced in vivo. Recently, we used this model to screen 400 herbal extracts and found 45 of them to be effective on reducing tau-GFP-induced neuronal death. One of the effective herbal extracts is the Tripterygium wilfordii stem extract. HPLC analysis and functional assay demonstrated that epicatechin (EC) is the major compound of Tripterygium wilfordii stem extract to decrease the neurotoxicity induced by tau-GFP. Using a luciferase reporter assay in the zebrafish, we confirmed that EC could activate Nrf2-dependent antioxidant responses to significantly increase the ARE-controlled expression of luciferase reporter gene. These data suggest that EC from the Tripterygium wilfordii stem extract could diminish tau-GFP-induced neuronal death through the activation of Nrf2.

Characterization of the Scutellaria barbata glycosyltransferase gene and its promoter.

The conversion of flavonoid aglycones to their glycosides by plant glycosyltransferases may affect a wide range of outcomes, including stability, solubility and bioavailability. Scutellaria barbata, rich in flavonoid glycosides, is widely used as a traditional Chinese herbal medicine. In this study, a flavonoid glycosyltransferase cDNA (SbUGT) and its promoter from S. barbata were cloned and characterized as a flavonoid glycosyltransferase using whole-cell biotransformation. Fragments of different lengths of the 5'-flanking region of the SbUGT gene were fused to the beta-glucuronidase (GUS) gene and analyzed with transgenic Arabidopsis plants using histochemical and fluorometric assays. GUS activity in transgenic plants carrying the SbP-850U construct (-850 to +86 relative to the transcription start site) displayed the highest level and was enhanced by salt and methyl jasmonate, similar to the expression patterns of the endogenous SbUGT. GUS activity disappeared when the promoter was deleted to -98, and deletion analyses indicated the existence of positive and negative regulatory element(s). Unexpectedly, plants carrying the construct SbP-102U (-102 to +86) exhibited strong GUS activity exclusively in the roots. Our experiments revealed that the specific expression is mediated by different promoter regions and the unique region driving root-preferred expression can be used as a root-specific promoter.

Authentication of medicinal herbs using PCR-amplified ITS2 with specific primers.

Different parts of medicinal herbs have long been used as traditional Chinese drugs for treating many diseases, whereas materials of similar morphology and chemical fingerprints are often misidentified. Analyses of sequence variations in the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) have become a valid method for authentication of medicinal herbs at the intergenic and interspecific levels. DNA extracted from processed materials is usually severely degraded or contaminated by microorganisms, thus generates no or unexpected PCR products. The goal of this study is to apply the ITS fragments selectively amplified with two designed primer sets for efficient and precise authentication of medicinal herbs. The designed primers led to an accurate PCR product of the specific region in ITS2, which was confirmed with DNA extracted from 55 processed medicinal herbs belonging to 48 families. Moreover, the selectively amplified ITS2 authenticated five sets of easily confusable Chinese herbal materials. The designed primers were proven to be suitable for a broad application in the authentication of herbal materials.