RESUMO
Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational abilities and cellular adaptation to environmental changes. Duplications of ribosomal protein (RP) genes are ubiquitous among organisms and are believed to affect cell function through paralog-specific regulation (e.g., by generating heterogeneous ribosomes) and/or gene dose amplification. However, direct evaluations of their impacts on cell function remain elusive due to the highly heterogeneous cellular RP pool. Here, we engineered a yeast with homogeneous 40S RP paralog compositions, designated homo-40S, by deleting the entire set of alternative duplicated genes encoding yeast 40S RP paralogs. Homo-40S displayed mild growth defects along with high sensitivity to the translation inhibitor paromomycin and a significantly increased stop codon readthrough. Moreover, doubling of the remaining RP paralogous genes in homo-40S rescued these phenotypes markedly, although not fully, compared to the wild-type phenotype, indicating that the dose of 40S RP genes together with the heterogeneity of the contents was vital for maintaining normal translational functionalities and growth robustness. Additional experiments revealed that homo-40S improved paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to generate fast-growing derivatives, highlighting the mutational robustness of engineered yeast to accommodate environmental and genetic changes. In summary, our work demonstrated that duplicated RP paralogs impart robustness and phenotypic plasticity through both gene dose amplification and paralog-specific regulation, paving the way for the direct study of ribosome biology through monotypic ribosomes with a homogeneous composition of specific RP paralogs.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas , Subunidades Ribossômicas Menores de Eucariotos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Engenharia Genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels â¼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2ß2γ2 hexamers in which the core catalytic α- and ß-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase. Comparison with UDP-glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases.
Assuntos
Lisossomos , Mucolipidoses , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Microscopia Crioeletrônica , Drosophila melanogaster , Lisossomos/química , Lisossomos/genética , Lisossomos/metabolismo , Mamíferos/metabolismo , Mucolipidoses/genética , Proteínas , Relação Estrutura-Atividade , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
During cellular translation of messenger RNAs by ribosomes, the translation apparatus sometimes pauses or stalls at the elongation and termination steps. With the exception of programmed stalling, which is usually used by cells for regulatory purposes, ribosomes stalled on mRNAs need to be terminated and recycled to maintain adequate translation capacity. Much ribosome stalling originates in aberrant mRNAs that lack a stop codon. Transcriptional errors, misprocessing of primary transcripts, and undesired mRNA cleavage all contribute to the formation of non-stop mRNAs. Ribosomes stalled at the 3' end of non-stop mRNAs do not undergo normal termination owing to the lack of specific stop-codon recognition by canonical peptide release factors at the A-site decoding centre. In bacteria, the transfer-messenger RNA (tmRNA)-SmpB-mediated trans-translation rescue system reroutes stalled ribosomes to the normal elongation cycle and translation termination. Two additional rescue systems, ArfA-RF2 (refs 13, 14, 15, 16) and ArfB (formerly known as YaeJ), are also present in many bacterial species, but their mechanisms are not fully understood. Here, using cryo-electron microscopy, we characterize the structure of the Escherichia coli 70S ribosome bound with ArfA, the release factor RF2, a short non-stop mRNA and a cognate P-site tRNA. The C-terminal loop of ArfA occupies the mRNA entry channel on the 30S subunit, whereas its N terminus is sandwiched between the decoding centre and the switch loop of RF2, leading to marked conformational changes in both the decoding centre and RF2. Despite the distinct conformation of RF2, its conserved catalytic GGQ motif is precisely positioned next to the CCA-end of the P-site tRNA. These data illustrate a stop-codon surrogate mechanism for ArfA in facilitating the termination of non-stop ribosomal complexes by RF2.
Assuntos
Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Códon de Terminação , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Modelos Moleculares , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/ultraestrutura , Ligação Proteica , Conformação Proteica , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Ribossomos/química , Ribossomos/ultraestruturaRESUMO
Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors, organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.
Assuntos
Microscopia Crioeletrônica , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Transporte Ativo do Núcleo Celular , Sequência de Bases , Domínio Catalítico , Núcleo Celular/química , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citoplasma/metabolismo , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , DNA Espaçador Ribossômico/metabolismo , DNA Espaçador Ribossômico/ultraestrutura , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/ultraestrutura , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Ligação Proteica , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Fúngico/ultraestrutura , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico/ultraestrutura , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestrutura , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Rotação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a bottleneck. Although cryo-focused ion beam (cryo-FIB) milling has emerged for large and flat cryo-lamella preparation, its application to tissue specimens remains challenging. Here, we report an integrated workflow, VHUT-cryo-FIB, for efficiently preparing frozen hydrated tissue lamella that can be readily used in subsequent cryo-ET studies. The workflow includes vibratome slicing, high-pressure freezing, ultramicrotome cryo-trimming and cryo-FIB milling. Two strategies were developed for loading cryo-lamella via a side-entry cryo-holder or an FEI AutoGrid. The workflow was validated by using various tissue specimens, including rat skeletal muscle, rat liver and spinach leaf specimens, and in situ structures of ribosomes were obtained at nanometer resolution from the spinach and liver samples.
Assuntos
Tomografia com Microscopia Eletrônica , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Congelamento , Íons , Substâncias MacromolecularesRESUMO
Many ribosome-interacting GTPases, with proposed functions in ribosome biogenesis, are also implicated in the cellular regulatory coupling between ribosome assembly process and various growth control pathways. EngA is an essential GTPase in bacteria, and intriguingly, it contains two consecutive GTPase domains (GD), being one-of-a-kind among all known GTPases. EngA is required for the 50S subunit maturation. However, its molecular role remains elusive. Here, we present the structure of EngA bound to the 50S subunit. Our data show that EngA binds to the peptidyl transferase center (PTC) and induces dramatic conformational changes on the 50S subunit, which virtually returns the 50S subunit to a state similar to that of the late-stage 50S assembly intermediates. Very interestingly, our data show that the two GDs exhibit a pseudo-two-fold symmetry in the 50S-bound conformation. Our results indicate that EngA recognizes certain forms of the 50S assembly intermediates, and likely facilitates the conformational maturation of the PTC of the 23S rRNA in a direct manner. Furthermore, in a broad context, our data also suggest that EngA might be a sensor of the cellular GTP/GDP ratio, endowed with multiple conformational states, in response to fluctuations in cellular nucleotide pool, to facilitate and regulate ribosome assembly.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Ligação ao GTP/química , Subunidades Ribossômicas Maiores de Bactérias/química , Microscopia Crioeletrônica , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Subunidades Ribossômicas Maiores de Bactérias/metabolismoRESUMO
BACKGROUND: Tea (Camellia sinensis) has long been consumed worldwide for its amazing flavor and aroma. Methyl jasmonate (MeJA), which acts as an effective elicitor among the plant kingdom, could mostly improve the quality of tea aroma by promoting flavor volatiles in tea leaves. Although a variety of volatile secondary metabolites that contribute to aroma quality have been identified, our understanding of the biosynthetic pathways of these compounds has remained largely incomplete. Therefore, information aboaut the transcriptome of tea leaves and, specifically, details of any changes in gene expression in response to MeJA, is required for a better understanding of the biological mechanisms of MeJA-mediated volatiles biosynthesis. Moreover, MeJA treatment could exaggerate the responses of secondary metabolites and some gene expression which offer a better chance to figure out the mechanism. RESULTS: The results of two-dimensional gas-chromatograph mass-spectrometry showed that the terpenoids content in MeJA-treated tea leaves increased, especially linalool, geraniol, and phenylethyl alcohol. More importantly, we carried out RNA-seq to identify the differentially expressed genes (DEGs) related to volatiles biosynthesis pathways induced by MeJA treatment (0 h, 12 h, 24 h and 48 h) in tea leaves. We identified 19245, 18614, 11890 DEGs respectively in the MeJA_12h, MeJA_24 h and MeJA_48 h samples. The α-Lenolenic acid degradation pathway was firstly responded resulting in activating the JA-pathway inner tea leaves, and the MEP/DOXP pathway significantly exaggerated. Notably, the expression level of jasmonate O-methyltransferase, which is associated with the central JA biosynthesis pathway, was increased by 7.52-fold in MeJA_24 h tea leaves. Moreover, the genes related to the terpenoid backbone biosynthesis pathway showed different expression patterns compared with the untreated leaves. The expression levels of 1-deoxy-D-xylulose-phosphate synthase (DXS), all-trans-nonaprenyl-diphosphate synthase, geranylgeranyl reductase, geranylgeranyl diphosphate synthase (type II), hydroxymethylglutaryl-CoA reductase and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase increased by approximately 2-4-fold. CONCLUSIONS: The results of two-dimension gas-chromatography mass-spectrometry analysis suggested that exogenous application of MeJA could induce the levels of volatile components in tea leaves, especially the geraniol, linalool and its oxides. Moreover, the transcriptome analysis showed increased expression of genes in α-Lenolenic acid degradation pathway which produced massive jasmonic acid and quickly activated holistic JA-pathway inner tea leaves, also the terpenoid backbones biosynthesis pathway was significantly affected after MeJA treatment. In general, MeJA could greatly activate secondary metabolism pathways, especially volatiles. The results will deeply increase our understanding of the volatile metabolites biosynthesis pathways of tea leaves in response to MeJA.
Assuntos
Camellia sinensis/genética , Camellia sinensis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Terpenos/metabolismo , Acetatos/farmacologia , Ciclopentanos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , Oxilipinas/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Análise de Sequência de DNA , Transcriptoma , Compostos Orgânicos Voláteis/metabolismoRESUMO
Ribosome assembly is a process fundamental for all cellular activities. The efficiency and accuracy of the subunit assembly are tightly regulated and closely monitored. In the present work, we characterized, both compositionally and structurally, a set of in vivo 50S subunit precursors (45S), isolated from a mutant bacterial strain. Our qualitative mass spectrometry data indicate that L28, L16, L33, L36 and L35 are dramatically underrepresented in the 45S particles. This protein spectrum shows interesting similarity to many qualitatively analyzed 50S precursors from different genetic background, indicating the presence of global rate-limiting steps in the late-stage assembly of 50S subunit. Our structural data reveal two major intermediate states for the 45S particles. Consistently, both states severally lack those proteins, but they also differ in the stability of the functional centers of the 50S subunit, demonstrating that they are translationally inactive. Detailed analysis indicates that the orientation of H38 accounts for the global conformational differences in these intermediate structures, and suggests that the reorientation of H38 to its native position is rate-limiting during the late-stage assembly. Especially, H38 plays an essential role in stabilizing the central protuberance, through the interaction with the 5S rRNA, and the correctly orientated H38 is likely a prerequisite for further maturation of the 50S subunit.
Assuntos
Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Microscopia Crioeletrônica , GTP Fosfo-Hidrolases/genética , Modelos Moleculares , RNA Ribossômico 23S/químicaRESUMO
For the manufacturing and sale of tea, rapid discrimination of overall quality grade is of great importance. However, present evaluation methods are time-consuming and labor-intensive. This study investigated the feasibility of combining advantages of near-infrared spectroscopy (NIRS) and electronic nose (E-nose) to assess the tea quality. We found that NIRS and E-nose models effectively identify taste and aroma quality grades, with the highest accuracies of 99.63% and 97.00%, respectively, by comparing different principal component numbers and classification algorithms. Additionally, the quantitative models based on NIRS predicted the contents of key substances. Based on this, NIRS and E-nose data were fused in the feature-level to build the overall quality evaluation model, achieving accuracies of 98.13%, 96.63% and 97.75% by support vector machine, K-nearest neighbors, and artificial neural network, respectively. This study reveals that the integration of NIRS and E-nose presents a novel and effective approach for rapidly identifying tea quality.
Assuntos
Camellia sinensis , Chá , Chá/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Nariz Eletrônico , Camellia sinensis/química , AlgoritmosRESUMO
Eukaryotic ribosome assembly is a highly orchestrated process that involves over two hundred protein factors. After early assembly events on nascent rRNA in the nucleolus, pre-60S particles undergo continuous maturation steps in the nucleoplasm, and prepare for nuclear export. Here, we report eleven cryo-EM structures of the nuclear pre-60S particles isolated from human cells through epitope-tagged GNL2, at resolutions of 2.8-4.3 Å. These high-resolution snapshots provide fine details for several major structural remodeling events at a virtual temporal resolution. Two new human nuclear factors, L10K and C11orf98, were also identified. Comparative structural analyses reveal that many assembly factors act as successive place holders to control the timing of factor association/dissociation events. They display multi-phasic binding properties for different domains and generate complex binding inter-dependencies as a means to guide the rRNA maturation process towards its mature conformation. Overall, our data reveal that nuclear assembly of human pre-60S particles is generally hierarchical with short branch pathways, and a few factors display specific roles as rRNA chaperones by confining rRNA helices locally to facilitate their folding, such as the C-terminal domain of SDAD1.
Assuntos
Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Modelos Moleculares , Ribossomos/química , Núcleo Celular/metabolismo , RNA Ribossômico/química , Proteínas Ribossômicas/metabolismoRESUMO
The type II AAA + ATPase Drg1 is a ribosome assembly factor, functioning to release Rlp24 from the pre-60S particle just exported from nucleus, and its activity in can be inhibited by a drug molecule diazaborine. However, molecular mechanisms of Drg1-mediated Rlp24 removal and diazaborine-mediated inhibition are not fully understood. Here, we report Drg1 structures in different nucleotide-binding and benzo-diazaborine treated states. Drg1 hexamers transits between two extreme conformations (planar or helical arrangement of protomers). By forming covalent adducts with ATP molecules in both ATPase domain, benzo-diazaborine locks Drg1 hexamers in a symmetric and non-productive conformation to inhibits both inter-protomer and inter-ring communication of Drg1 hexamers. We also obtained a substrate-engaged mutant Drg1 structure, in which conserved pore-loops form a spiral staircase to interact with the polypeptide through a sequence-independent manner. Structure-based mutagenesis data highlight the functional importance of the pore-loop, the D1-D2 linker and the inter-subunit signaling motif of Drg1, which share similar regulatory mechanisms with p97. Our results suggest that Drg1 may function as an unfoldase that threads a substrate protein within the pre-60S particle.
Assuntos
Adenosina Trifosfatases , Chaperonas Moleculares , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/metabolismo , Chaperonas Moleculares/metabolismo , Conformação Molecular , Domínios Proteicos , Trifosfato de Adenosina/metabolismoRESUMO
The lateral segregation of membrane constituents into functional microdomains, conceptually known as lipid raft, is a universal organization principle for cellular membranes in both prokaryotes and eukaryotes. The widespread Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) family proteins are enriched in functional membrane microdomains at various subcellular locations, and therefore were hypothesized to play a scaffolding role in microdomain formation. In addition, many SPFH proteins are also implicated in highly specific processes occurring on the membrane. However, none of these functions is understood at the molecular level. Here we report the structure of a supramolecular complex that is isolated from bacterial membrane microdomains and contains two SPFH proteins (HflK and HflC) and a membrane-anchored AAA+ protease FtsH. HflK and HflC form a circular 24-mer assembly, featuring a laterally segregated membrane microdomain (20 nm in diameter) bordered by transmembrane domains of HflK/C and a completely sealed periplasmic vault. Four FtsH hexamers are embedded inside this microdomain through interactions with the inner surface of the vault. These observations provide a mechanistic explanation for the role of HflK/C and their mitochondrial homologs prohibitins in regulating membrane-bound AAA+ proteases, and suggest a general model for the organization and functionalization of membrane microdomains by SPFH proteins.
Assuntos
Bactérias , Microdomínios da Membrana , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismoRESUMO
Plastic baskets are commonly used as containers for fresh tea leaves during storage and transport after harvest. Nevertheless, there are significant challenges in controlling the core temperature of the basket since fresh tea leaves still maintain a certain degree of respiration after being harvested, with extremely high temperatures being the major factor for the color change of fresh tea leaves. A numerical model was developed to improve the temperature control of the plastic basket, by which the influence of different structural parameters on the core temperature in the plastic baskets with fresh tea leaves was analyzed. The accuracy of the model in predicting airflow and temperature distributions was validated against experimental data. The maximum RMSE was 1.158 °C and the maximum MRE was 5.410% between the simulated and test temperature value. The maximum deviation between the simulated velocity and test velocity was 0.11 m/s, the maximum RE was 29.05% and the maximum SD was 0.024. The results show that a plastic basket with a ventilation duct efficiently decreased the temperature of the fresh tea leaves and significantly affected the heat transfer between the fresh tea leaves and the ambient air compared to the plastic basket without a ventilation duct. Furthermore, the effect on the heat transfer was further expanded by the use of a plastic basket with a ventilation duct when the plastic baskets were stacked. The maximum temperature differences were 0.52 and 0.40 according to the stacked and single-layer products, respectively. The ambient temperature and the bulk density of the fresh tea leaves have a significant influence on the core temperature.
RESUMO
The study was aim to investigate the effects of grafting on volatile compounds and sensory quality of black tea. Seven groups of black tea were prepared from one nongrafted tea tree "Yinghong9 (YJ)" and six grafted tea trees by grafting scion of "YingHong9" on different rootstocks. Sensory analysis indicated marked/slight variations among seven samples, among which, the one grafting on HuangZhiXiangDanCong (HZX) stood out with floral and fruity aroma. The result of chemometrics analysis suggested various effects on compounds caused by different rootstocks. A total of 38 differential compounds were identified, showing mainly quantitative variations, with 36 being identified in all samples. The significant higher contents of volatiles, such as geraniol, phenylethyl alcohol, (E)-nerolidol, decanal, and linalool oxides, in HZX compared with YJ were observed, which explained why floral and fruity aroma stood out among the whole aroma profile of HZX. Both results of sensory and instrumental analysis suggested certain correlation between compound variations and aroma characteristics. Moreover, different rootstocks influenced the aroma quality in different ways. PRACTICAL APPLICATION: In conclusion, the study illuminates the various effects of grafting on the volatile compounds and aroma quality, which enlightens the possibility of changing aroma quality of black tea by grafting scions on different rootstocks. And thus, it can help guide the practical production when cultivating new varieties.
Assuntos
Agricultura/métodos , Camellia sinensis/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas/métodos , Odorantes/análise , Chá/química , Humanos , Raízes de Plantas/crescimento & desenvolvimento , Olfato , Compostos Orgânicos Voláteis/análiseRESUMO
O(6)-methylguanine-DNA methyltransferase is one of the rare proteins to directly remove alkylating agents in the human DNA direct reversal repair pathway. Its two common single-nucleotide polymorphisms, Leu84Phe and Ile143Val, had previously been identified to contribute to susceptibility of cancer. However, there are conflicting results in studies on the association of the two polymorphisms with cancer. Therefore, we conducted a meta-analysis to clarify the paradox with a large collected sample (13,069 cancer patients and 20,290 controls). We found significant association between the T allele (84Phe) and cancer risk, under the recessive genetic model [P = 0.023, odds ratio (OR) = 1.251, 95% confidence interval (CI) 1.031-1.517, P(heterogeneity) = 0.270], TT versus CC comparison (P = 0.035, OR = 1.239, 95% CI 1.015-1.511, P(heterogeneity) = 0.225) and TT versus CT comparison (P = 0.007, OR = 1.292, 95% CI 1.071-1.559, P(heterogeneity) = 0.374), using the random-effect model. In the ethnicity subgroup analysis, a significant association with cancer among Caucasians was found under the recessive genetic model, homozygote comparison and TT versus TC comparison. In the tumour sites subgroup analysis, only the protective effects of Leu84Phe polymorphism were found in colorectal cancer, under CT versus CC comparison. No significant association between the G allele of Ile143Val and cancer risk was found. The G allele showed an increased lung cancer risk under the dominant genetic model and AG versus AA comparison in all Hardy-Weinberg equilibrium subjects, only when the fixed-effect model was used. However, it was insignificant in the random-effect model.
Assuntos
Neoplasias/genética , O(6)-Metilguanina-DNA Metiltransferase/genética , Polimorfismo de Nucleotídeo Único/genética , Genes Recessivos , Humanos , Modelos Genéticos , Neoplasias/etnologia , Razão de Chances , Fatores de Risco , População BrancaRESUMO
Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk. Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors.
Assuntos
Núcleo Celular/metabolismo , Modelos Moleculares , RNA Ribossômico 5S/ultraestrutura , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Microscopia Crioeletrônica , Humanos , RNA Ribossômico 5S/isolamento & purificação , RNA Ribossômico 5S/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestruturaRESUMO
The Sec61/SecY channel allows the translocation of many proteins across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. In bacteria, most secretory proteins are transported post-translationally through the SecY channel by the SecA ATPase. How a polypeptide is moved through the SecA-SecY complex is poorly understood, as structural information is lacking. Here, we report an electron cryo-microscopy (cryo-EM) structure of a translocating SecA-SecY complex in a lipid environment. The translocating polypeptide chain can be traced through both SecA and SecY. In the captured transition state of ATP hydrolysis, SecA's two-helix finger is close to the polypeptide, while SecA's clamp interacts with the polypeptide in a sequence-independent manner by inducing a short ß-strand. Taking into account previous biochemical and biophysical data, our structure is consistent with a model in which the two-helix finger and clamp cooperate during the ATPase cycle to move a polypeptide through the channel.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Canais de Translocação SEC/metabolismo , Adenosina Trifosfatases/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Cristalização , Escherichia coli , Geobacillus/metabolismo , Modelos Moleculares , Conformação Proteica , Transporte Proteico , Canais de Translocação SEC/genética , Proteínas SecARESUMO
To investigate the effects of grafting on non-volatile metabolites in tea, non-targeted metabolomic analyses of fresh leaves were performed on the basis of ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF/MS). One non-grafted YingHong No. 9 and four grafted tea [grafting scion YingHong No. 9 on four different rootstocks, BaiMao No. 2 (BM2), BaiYeDanCong (BY), HeiYeShuiXian (HY), and WuLingHong (WLH)] were chosen as materials. In total, 32 differential metabolites were identified, including phenolic acids, flavan-3-ols, dimeric catechins, flavonol and flavonol/flavone glycosides, etc. Partial least squares discrimination analysis and hierarchical cluster analysis showed various effects of different rootstocks on metabolites. Thereinto, rootstocks of WLH and BY showed extremely outstanding performance in up- and downregulating these metabolites, respectively. Differential metabolites were enriched into three crucial pathways, including biosynthesis of phenylpropanoids, flavonoid biosynthesis, and flavone and flavonol biosynthesis, which might influence the quality of tea. This study provides a theoretical basis for grafting-related variations of non-volatile metabolites in fresh tea leaves.
Assuntos
Camellia sinensis/química , Extratos Vegetais/química , Folhas de Planta/química , Camellia sinensis/metabolismo , Cromatografia Líquida de Alta Pressão , Metabolômica , Melhoramento Vegetal , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Espectrometria de Massas em TandemRESUMO
Two large protein-cofactor complexes, photosystem I and photosystem II, are the central components of photosynthesis in the thylakoid membranes. Here, we report the 2.37-Å structure of a tetrameric photosystem I complex from a heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Four photosystem I monomers, organized in a dimer of dimer, form two distinct interfaces that are largely mediated by specifically orientated polar lipids, such as sulfoquinovosyl diacylglycerol. The structure depicts a more closely connected network of chlorophylls across monomer interfaces than those seen in trimeric PSI from thermophilic cyanobacteria, possibly allowing a more efficient energy transfer between monomers. Our physiological data also revealed a functional link of photosystem I oligomerization to cyclic electron flow and thylakoid membrane organization.