Comparative Metabolomics and Transcriptome Analysis Reveal the Fragrance-Related Metabolite Formation in Phoebe zhennan Wood.

Nanmu (Phoebe zhennan) has a unique fragrance and is a high-quality tree species for forest conservation. The types and contents of volatile compounds in different tissues of nanmu wood are different, and the study of its volatile metabolites can help us to understand the source of its fragrance and functions. In order to explore the metabolites related to the wood fragrance of nanmu and to find out the unique volatile substances in the heartwood, gas chromatography-mass spectrometry (GC-MS) was performed to analyze the non-targeted metabolomics in five radial tissues from the sapwood to the heartwood of nanmu. A total of 53 volatile metabolites belonging to 11 classes were detected in all tissues, including terpenes, aromatic hydrocarbons, organoheterocyclics, phenols, esters, organic acids, alcohols, alkaloids, alkane, indoles derivatives, and others. And most of the volatile metabolites were identified for the first time in nanmu wood. Among them, terpenes and aromatic hydrocarbons were the main volatile components. In addition, 22 differential metabolites were screened from HW and SW, HW, and TZ via metabolomic analysis. Among these DAMs, three volatile metabolites (cadinene, a sesquiterpenoid; p-cymene, a monoterpenoid; 1,3,5-triisopropylbenzene, an aromatic hydrocarbon) contributed heavily to the characteristic fragrance of the heartwood. Additionally, the expression of transcripts showed that the unigenes in the terpenoid biosynthesis pathway were especially up-regulated in the SW. Therefore, we speculated that fragrance-related metabolites were synthesized in SW and then deposited in heartwood during sapwood transformed to heartwood. The expression levels of transcription factors (e.g., WRKY, C2H2, NAC) acted as the major regulatory factors in the synthesis of terpenoid. The results lay the foundations for further studies on the formation mechanism of fragrance components in nanmu wood and also provide a reference for the further development and utilization of nanmu wood.

Integrated Transcriptomic, Metabolomic, and Physiological Analyses Reveal New Insights into Fragrance Formation in the Heartwood of Phoebe hui.

Phoebe hui is an extremely valuable tree that is the main source of the fragrant golden-thread nanmu wood. Although the fragrance of wood has been investigated in several trees, the potential substances and gene regulation mechanisms that are involved in fragrance formation are poorly understood. Here, three radial tissues, sapwood (SW), heartwood (HW), and the transition zone (TZ) in between them, were compared via integrative physiological, volatile-metabolomic, and transcriptomic analyses to identify the key metabolites and regulatory mechanisms involved in fragrance formation. During heartwood formation, gradual starch grain loss was accompanied by the deposition of lipids and extractives in the cell lumen. Extracts of terpenoids were synthesized and accumulated in the heartwood, including monoterpenoids (limonene and p-cymene) and sesquiterpenes (cubebene and guaiadiene); these were identified as being closely related to the special fragrance of the wood. Additionally, the expression of transcripts showed that the genes related to primary metabolism were specifically upregulated in the SW, whereas genes annotated in terpenoid biosynthesis were specifically upregulated in the HW. Therefore, we speculated that terpenoid biosynthesis occurs in situ in the HW via the HW formation model of Type-III (Santalum) using the precursors that were produced by primary metabolism in the SW. The expression levels of transcription factors (e.g., MYB, WRKY, and C2H2) acted as the major regulatory factors in the synthesis of terpenoids. Our results explain the special fragrance in P. hui and broaden the current knowledge of the regulatory mechanisms of fragrance formation. This work provides a framework for future research that is focused on improving wood quality and value.

Integrative Metabolomic and Transcriptomic Analysis Reveals the Mechanism of Specific Color Formation in Phoebe zhennan Heartwood.

Nanmu (Phoebe zhennan) is an extremely valuable tree plant that is the main source of famous "golden-thread nanmu" wood. The potential metabolites and gene regulation mechanisms involved in golden thread formation are poorly understood, even though the color change from sapwood to heartwood has been investigated in several tree plants. Here, five radial tissues from sapwood to heartwood were compared via integrative metabolomic and transcriptomic analysis to reveal the secondary metabolites and molecular mechanisms involved in golden thread formation. During heartwood formation, gradual starch grain loss is accompanied by the cell lumen deposition of lipids and color-related extractives. Extractives of 20 phenylpropanoids accumulated in heartwood, including cinnamic acids and derivatives, coumarin acid derivatives, and flavonoids, which were identified as being closely related to the golden thread. Phenylpropanoids co-occurring with abundant accumulated metabolites of prenol lipids, fatty acyls, steroids, and steroid derivatives may greatly contribute to the characteristics of golden thread formation. Additionally, the expression of nine genes whose products catalyze phenylpropanoid and flavonoids biosynthesis was upregulated in the transition zone, then accumulated and used to color the heartwood. The expression levels of transcription factors (e.g., MYB, bHLH, and WRKY) that act as the major regulatory factors in the synthesis and deposition of phenylpropanoid and flavonoids responsible for golden thread formation were also higher than in sapwood. Our results not only explain golden thread formation in nanmu, but also broaden current knowledge of special wood color formation mechanisms. This work provides a framework for future research focused on improving wood color.

An HMGA2-p62-ERα axis regulates uterine leiomyomas proliferation.

Uterine leiomyomas (ULM) are a major public health issue contributing to high morbidity and poor pregnancy outcomes. However, its molecular pathogenesis is poorly understood. HMGA2-ULM is the second major subtype of human ULM and associates with large sizes, fast-growth, and high percentages of estrogen receptor α (ERα). As altered ERα expression plays a distinct role in ULM growth, here, we investigate a regulatory mechanism driving ULM growth via HMGA2 and ERα. We reveal a positive correlation of HMGA2 with ERα protein and demonstrate that HMGA2 promotes ULM cells proliferation via ERα. In addition, autophagy pathway and p62/SQSTM1 (a selective autophagy receptor) are found to participate in the regulation of HMGA2 and ERα. Moreover, HMGA2 suppresses the transcription of p62 by binding to its promoter, meanwhile, p62 interacts with ERα, and inhibition of p62 increases ERα expression and enhances cell viability in ULM, suggesting a novel mechanism of the HMGA2-p62-ERα axis in ULM proliferation. Notably, rapamycin, a familiar autophagy agonist, reduces ERα levels and the proliferation ability of ULM cells. This study demonstrates a causal role of the HMGA2-p62-ERα axis in preventing autophagy and increasing ERα expression in HMGA2-ULM. Therefore, blocking HMGA2-p62-ERα axis and targeting autophagy pathway establish a roadmap toward HMGA2-ULM medical treatment.

Crystal structure of human lysyl oxidase-like 2 (hLOXL2) in a precursor state.

Lysyl oxidases (LOXs), a type of copper- and lysyl tyrosylquinone (LTQ) -dependent amine oxidase, catalyze the oxidative deamination of lysine residues of extracellular matrix (ECM) proteins such as elastins and collagens and generate aldehyde groups. The oxidative deamination of lysine represents the foundational step for the cross-linking of elastin and collagen and thus is crucial for ECM modeling. Despite their physiological significance, the structure of this important family of enzymes remains elusive. Here we report the crystal structure of human lysyl oxidase-like 2 (hLOXL2) at 2.4-Å resolution. Unexpectedly, the copper-binding site of hLOXL2 is occupied by zinc, which blocks LTQ generation and the enzymatic activity of hLOXL2 in our in vitro assay. Biochemical analysis confirms that copper loading robustly activates hLOXL2 and supports LTQ formation. Furthermore, the LTQ precursor residues in the structure are distanced by 16.6 Å, corroborating the notion that the present structure may represent a precursor state and that pronounced conformational rearrangements would be required for protein activation. The structure presented here establishes an important foundation for understanding the structure-function relationship of LOX proteins and will facilitate LOX-targeting drug discovery.

Transcriptome analysis of terpenoid biosynthetic genes and simple sequence repeat marker screening in Eucommia ulmoides.

Trans-polyisoprene rubber is produced in the tissues of leaves, bark, and fruit of Eucommia ulmoides and is considered an important energy source. Transcript profiles of two tissues from E. ulmoides cv. Qinzhong No. 3, leaf and fruit, were analysed using the Illumina HiSeq 2000 system. In total, 104 million clean reads were obtained and assembled into 58,863 unigenes. Through gene functional classification, 28,091 unigenes (47.72%) were annotated and 65 unigenes have been hypothesized to encode proteins involved in terpenoid biosynthesis. In addition, 10,041 unigenes were detected as differentially expressed unigenes, and 29 of them were putatively related to terpenoid biosynthesis. The synthesis of trans-polyisoprene rubbers in E. ulmoides was hypothesised to be dominated by the mevalonate pathway. Farnesyl diphosphate synthase 2 (FPPS2) was considered a key component in the biosynthesis of trans-polyprenyl diphosphate. Rubber elongation factor 3 (REF3) might be involved in stabilising the membrane of rubber particles in E. ulmoides. To date, 351 simple sequence repeats (SSRs) were validated as polymorphisms from eight E. ulmoides plants (two parent plants and six F1 individuals), and these could act as molecular markers for genetic map density increase and breeding improvement of E. ulmoides.

Structural and mechanistic basis for the inhibition of Escherichia coli RNA polymerase by T7 Gp2.

The T7 phage-encoded small protein Gp2 is a non-DNA-binding transcription factor that interacts with the jaw domain of the Escherichia coli (Ec) RNA polymerase (RNAp) ß' subunit and inhibits transcriptionally proficient promoter-complex (RPo) formation. Here, we describe the high-resolution solution structure of the Gp2-Ec ß' jaw domain complex and show that Gp2 and DNA compete for binding to the ß' jaw domain. We reveal that efficient inhibition of RPo formation by Gp2 requires the amino-terminal σ(70) domain region 1.1 (R1.1), and that Gp2 antagonizes the obligatory movement of R1.1 during RPo formation. We demonstrate that Gp2 inhibits RPo formation not just by steric occlusion of the RNAp-DNA interaction but also through long-range antagonistic effects on RNAp-promoter interactions around the RNAp active center that likely occur due to repositioning of R1.1 by Gp2. The inhibition of Ec RNAp by Gp2 thus defines a previously uncharacterized mechanism by which bacterial transcription is regulated by a viral factor.

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx.

HBx is a hepatitis B virus protein that is required for viral infectivity and replication. Anti-apoptotic Bcl-2 family members are thought to be among the important host targets of HBx. However, the structure and function of HBx are poorly understood and the molecular mechanism of HBx-induced carcinogenesis remains unknown. In this study, we report biochemical and structural characterization of HBx. The recombinant HBx protein contains metal ions, in particular iron and zinc. A BH3-like motif in HBx (residues 110-135) binds Bcl-2 with a dissociation constant of ∼193 µM, which is drastically lower than that for a canonical BH3 motif from Bim or Bad. Structural analysis reveals that, similar to other BH3 motifs, the BH3-like motif of HBx adopts an amphipathic α-helix and binds the conserved BH3-binding groove on Bcl-2. Unlike the helical Bim or Bad BH3 motif, the C-terminal portion of the bound HBx BH3-like motif has an extended conformation and makes considerably fewer interactions with Bcl-2. These observations suggest that HBx may modulate Bcl-2 function in a way that is different from that of the classical BH3-only proteins.

Dynamic Changes in Metabolite Accumulation and the Transcriptome during Leaf Growth and Development in Eucommia ulmoides.

Eucommia ulmoides Oliver is widely distributed in China. This species has been used mainly in medicine due to the high concentration of chlorogenic acid (CGA), flavonoids, lignans, and other compounds in the leaves and barks. However, the categories of metabolites, dynamic changes in metabolite accumulation and overall molecular mechanisms involved in metabolite biosynthesis during E. ulmoides leaf growth and development remain unknown. Here, a total of 515 analytes, including 127 flavonoids, 46 organic acids, 44 amino acid derivatives, 9 phenolamides, and 16 vitamins, were identified from four E. ulmoides samples using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) (for widely targeted metabolites). The accumulation of most flavonoids peaked in growing leaves, followed by old leaves. UPLC-MS analysis indicated that CGA accumulation increased steadily to a high concentration during leaf growth and development, and rutin showed a high accumulation level in leaf buds and growing leaves. Based on single-molecule long-read sequencing technology, 69,020 transcripts and 2880 novel loci were identified in E. ulmoides. Expression analysis indicated that isoforms in the flavonoid biosynthetic pathway and flavonoid metabolic pathway were highly expressed in growing leaves and old leaves. Co-expression network analysis suggested a potential direct link between the flavonoid and phenylpropanoid biosynthetic pathways via the regulation of transcription factors, including MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic/helix-loop-helix). Our study predicts dynamic metabolic models during leaf growth and development and will support further molecular biological studies of metabolite biosynthesis in E. ulmoides. In addition, our results significantly improve the annotation of the E. ulmoides genome.

Anomalous Nernst Effect in the Dirac Semimetal Cd_{3}As_{2}.

Dirac and Weyl semimetals display a host of novel properties. In Cd_{3}As_{2}, the Dirac nodes lead to a protection mechanism that strongly suppresses backscattering in a zero magnetic field, resulting in ultrahigh mobility (∼10^{7} cm^{2} V^{-1} s^{-1}). In an applied magnetic field, an anomalous Nernst effect is predicted to arise from the Berry curvature associated with the Weyl nodes. We report the observation of a large anomalous Nernst effect in Cd_{3}As_{2}. Both the anomalous Nernst signal and transport relaxation time τ_{tr} begin to increase rapidly at ∼50 K. This suggests a close relation between the protection mechanism and the anomalous Nernst effect. In a field, the quantum oscillations of bulk states display a beating effect, suggesting that the Dirac nodes split into Weyl states, allowing the Berry curvature to be observed as an anomalous Nernst effect.

Ultrahigh mobility and giant magnetoresistance in the Dirac semimetal Cd3As2.

Dirac and Weyl semimetals are 3D analogues of graphene in which crystalline symmetry protects the nodes against gap formation. Na3Bi and Cd3As2 were predicted to be Dirac semimetals, and recently confirmed to be so by photoemission experiments. Several novel transport properties in a magnetic field have been proposed for Dirac semimetals. Here, we report a property of Cd3As2 that was unpredicted, namely a remarkable protection mechanism that strongly suppresses backscattering in zero magnetic field. In single crystals, the protection results in ultrahigh mobility, 9 × 10(6) cm(2) V(-1) s(-1) at 5 K. Suppression of backscattering results in a transport lifetime 10(4) times longer than the quantum lifetime. The lifting of this protection by the applied magnetic field leads to a very large magnetoresistance. We discuss how this may relate to changes to the Fermi surface induced by the applied magnetic field.

Composite Icosahedron/Cube Endohedral Clusters in Rh2Cd15.

We present Rh2Cd15, the first binary compound in the Rh-Cd system. It is based on transition-metal-embedded (Rh@Cd11 and Rh@Cd12) endohedral Cd clusters that are single- and double-capped IC10 composite icosahedra/cubes. We demonstrate the structural connections between the clusters. On the basis of the analysis of atomic interactions and electron counting, 50 electrons per Rh2Cd15 is postulated to be the boundary between bonding and antibonding interactions. This understanding is supported by electronic structure calculations showing that the electron count for Rh2Cd15 (48 electrons per Rh2Cd15) is located close to a deep pseudogap in the electronic density of states at 50 electrons per formula unit, which we postulate is an important factor in determining the new compound's stability.

A molecular genetic linkage map of Eucommia ulmoides and quantitative trait loci (QTL) analysis for growth traits.

Eucommia ulmoides is an economically important tree species for both herbal medicine and organic chemical industry. Effort to breed varieties with improved yield and quality is limited by the lack of knowledge on the genetic basis of the traits. A genetic linkage map of E. ulmoides was constructed from a full-sib family using sequence-related amplified polymorphism, amplified fragment length polymorphism, inter-simple sequence repeat and simple sequence repeat markers. In total, 706 markers were mapped in 25 linkage groups covering 2133 cM. The genetic linkage map covered approximately 89% of the estimated E. ulmoides genome with an average of 3.1 cM between adjacent markers. The present genetic linkage map was used to identify quantitative trait loci (QTL) affecting growth-related traits. Eighteen QTLs were found to explain 12.4%-33.3% of the phenotypic variance. This genetic linkage map provides a tool for marker-assisted selection and for studies of genome in E. ulmoides.

Optimization-based adaptive trajectory tracking controller design of self-balanced vehicle with asymptotic prescribed performance.

To handle with the nonlinear external disturbances and unmodeled dynamics of self-balanced vehicle (SBV), a novel adaptive trajectory tracking controller based on asymptotic prescribed performance is proposed. First, a velocity planner based on kinematic is constructed to control the velocity signal to improve the motion stability of SBV. Second, the prescribed performance function (PPF) is designed to prescribe transient-state and steady-state performances (TSP). Afterwards, an optimization-based predictive control (OPC) is proposed for accurate trajectory tracking of SBV. Furthermore, a modified radial basis function neural network (RBFNN) approximator is developed to compensate the unmodeled dynamics and the nonlinear external disturbances of the SBV. The overall system stability is proved with the help of Lyapunov theorem. Finally, the tracking performance and anti-interference robustness of the proposed control method are verified by comparative numerical simulations.

Evaluation of thyroid-disrupting effects of bisphenol F and bisphenol S on zebrafish (Danio rerio) using anti-transthyretin monoclonal antibody-based immunoassays.

The thyroid disrupting chemicals (TDCs) have raised great concerns due to their adverse impacts on thyroid hormones (THs). In this study, we investigated the thyroid-disrupting effects of bisphenol F (BPF) and bisphenol S (BPS), two major BPA substitutes, on adult zebrafish (Danio rerio). Firstly, anti-transthyretin (TTR) monoclonal antibody (anti-TTR mAb) was prepared and used to establish an indirect ELISA, which had a working range of 15.6∼1000 ng/mL of a detection limit of 6.1 ng/mL. The immunoassays based on anti-TTR mAb showed that exposure to BPF (10 and 100 µg/L) and BPS (100 µg/L) significantly elevated the levels of TTR protein in the plasma, liver, and brain tissues. Moreover, immunofluorescence showed that 100 µg/L BPF and BPS induced the production of TTR protein in liver and brain tissues. In addition, BPF and BPS increased THs levels and damaged thyroid tissue structure in adult female zebrafish. Especially, 100 µg/L BPF significantly increased T4 and T3 levels by 2.05 and 1.14 times, and induced pathological changes of thyroid follicles. The changes in the expression levels of genes involved in the hypothalamus-pituitary-thyroid (HPT) axis further illustrated that BPF and BPS had significant adverse effects on THs homeostasis and thyroid function in zebrafish. Therefore, TTR immunoassays could be used for the evaluation of thyroid-disrupting effects in fish and BPF exhibited greater disruption than BPS.

Nanoplastics increase the adverse impacts of lead on the growth, morphological structure and photosynthesis of marine microalga Platymonashelgolandica.

Nanoplastics and heavy metals are common pollutants in coastal environments with high concerns, but their joint ecological risk to marine primary productivity remains unclear. In this study, the effects of 7, 70, 700 µg/L lead (Pb) single exposure and in combination with 200 µg/L polystyrene nanoplastics (NPs, 70 nm) on marine microalga Platymonas helgolandica were investigated. Pb single exposure induced a dose-dependent inhibition on the growth of P. helgolandica, which was associated with the reduced photosynthetic efficiency and nutrient accumulation. Compared to Pb single exposure, the addition of NPs significantly reduced the photosynthetic efficiency and aggravated the damage to cell structure. Reduced esterase activity and increased membrane permeability also indicated that NPs exacerbated the adverse effects of Pb on P. helgolandica. Thus, co-exposure to NPs and Pb induced more severe impacts on marine microalgae, suggesting that the joint ecological risk of NPs and heavy metals to marine primary productivity merits more attention.

Development and application of bisphenol S electrochemical immunosensor and iridium oxide nanoparticle-based lateral flow immunoassay.

Bisphenol S (BPS) is a common pollutant in the environment and has posed a potential threat to aquatic animals and human health. To accurately assess the pollution level and ecological risk of BPS, there is an urgent need to establish simple and sensitive detection methods for BPS. In this study, BPS complete antigen was successfully prepared by introducing methyl 4-bromobutyrate and coupling bovine serum albumin (BSA). The monoclonal antibody against BPS (anti-BPS mAb) with high affinity (1: 256,000) was developed based on the BPS complete antigen, which showed low cross-reactivity with BPS structural analogues. Then, an electrochemical immunosensor was constructed to detect BPS using multi-walled carbon nanotubes and gold nanoflower composites as signal amplification elements and using anti-BPS mAb as the probe. The electrochemical immunosensor had a linear range from 1 to 250 ng⋅mL-1 and a limit of detection (LOD) down to 0.6 ng⋅mL-1. Additionally, a more stable and sensitive lateral flow immunoassay (LFIA) for BPS was developed based on iridium oxide nanoparticles, with a visual detection limit of 1 ng⋅mL-1, which was 10 times lower than that of classical Au-NPs LFIA. After evaluation of their stability and specificity, the reliability of these two methods were further validated by measuring BPS concentrations in the water and fish tissues. Thus, this study provides sensitive, robust and rapid methods for the detection of BPS in the environment and organisms, which can provide a methodological reference for monitoring environmental contaminants.

Micro-/nano-plastics as vectors of heavy metals and stress response of ciliates using transcriptomic and metabolomic analyses.

The escalating presence of microplastics and heavy metals in marine environments significantly jeopardizes ecological stability and human health. Despite this, research on the combined effects of microplastics/nanoplastics (MPs/NPs) and heavy metals on marine organisms remains limited. This study evaluated the impact of two sizes of polystyrene beads (approximately 2 µm and 200 nm) combined with cadmium (Cd) on the ciliate species Euplotes vannus. Results demonstrated that co-exposure of MPs/NPs and Cd markedly elevated reactive oxygen species (ROS) levels in ciliates while impairing antioxidant enzyme activities, thus enhancing oxidative damage and significantly reducing carbon biomass in ciliates. Transcriptomic profiling indicated that co-exposure of MPs/NPs and Cd potentially caused severe DNA damage and protein oxidation, as evidenced by numerous differentially expressed genes (DEGs) associated with mismatch repair, DNA replication, and proteasome function. Integrated transcriptomic and metabolomic analysis revealed that DEGs and differentially accumulated metabolites (DAMs) were significantly enriched in the TCA cycle, glycolysis, tryptophan metabolism, and glutathione metabolism. This suggests that co-exposure of MPs/NPs and Cd may reduce ciliate abundance and carbon biomass by inhibiting energy metabolism and antioxidant pathways. Additionally, compared to MPs, the co-exposure of NPs and Cd exhibited more severe negative effects due to the larger specific surface area of NPs, which can carry more Cd. These findings provide novel insights into the toxic effects of MPs/NPs and heavy metals on protozoan ciliates, offering foundational data for assessing the ecological risks of heavy metals exacerbated by MPs/NPs.

Identification and variation analysis of the composition and content of essential oil and fragrance compounds in Phoebe zhennan wood at different tree ages.

Wood essential oil and wood products with special fragrances are high value-added forest products. Despite the availability of essential oil and volatile organic compounds (VOCs) from Phoebe zhennan wood, their variation and dependence on tree age have not been examined. After essential oil extraction and wood processing, the yields and compositions of essential oils and VOCs in wood from P. zhennan trees of different ages (10a, 30a, and 80a) were determined. The yield of essential oil from 30a wood was significantly greater than that from 10a and 80a wood. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) revealed 672 and 41 volatile compounds, respectively, in the essential oil and wood, the majority of which exhibited large fluctuations in relative content and composition depending on tree age. Sesquiterpenoids, fatty acids and conjugates may greatly contribute to the main components of essential oil from wood. Almost all major sesquiterpenoid compounds, such as caryophyllene α-oxide, eudesmo, and cubebene, were identified in the essential oils from the 30a and 80a wood, and their relative contents were much greater than those in the 10a wood. The main components of the wood fragrance were sesquiterpenoids. The types and relative contents of sesquiterpenoids from wood increased with tree age. These results suggest that choosing wood from trees of a suitable age will significantly improve the efficiency of wood utilization.

T7 phage protein Gp2 inhibits the Escherichia coli RNA polymerase by antagonizing stable DNA strand separation near the transcription start site.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the host RNA polymerase (RNAP)--a multi-subunit enzyme responsible for gene transcription--by a small ( approximately 7 kDa) phage-encoded protein called Gp2. Gp2 is also a potent inhibitor of E. coli RNAP in vitro. Here we describe the first atomic resolution structure of Gp2, which reveals a distinct run of surface-exposed negatively charged amino acid residues on one side of the molecule. Our comprehensive mutagenesis data reveal that two conserved arginine residues located on the opposite side of Gp2 are important for binding to and inhibition of RNAP. Based on a structural model of the Gp2-RNAP complex, we propose that inhibition of transcription by Gp2 involves prevention of RNAP-promoter DNA interactions required for stable DNA strand separation and maintenance of the "transcription bubble" near the transcription start site, an obligatory step in the formation of a transcriptionally competent promoter complex.