ABSTRACT
Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes1,2. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)3,4. ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes5,6. Here we report the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5'-phosphate-binding pocket. The overall conformation of Dcr-2-Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2-Loqs-PD.
Subject(s)
Cryoelectron Microscopy , Drosophila Proteins , Drosophila melanogaster , RNA Helicases , RNA, Double-Stranded , RNA, Small Interfering , RNA-Binding Proteins , Ribonuclease III , Adenosine Triphosphate , Animals , Binding Sites , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Drosophila Proteins/ultrastructure , Phosphates/metabolism , Protein Conformation , RNA Helicases/chemistry , RNA Helicases/metabolism , RNA Helicases/ultrastructure , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/metabolism , RNA, Double-Stranded/ultrastructure , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , RNA, Small Interfering/ultrastructure , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/ultrastructure , Ribonuclease III/chemistry , Ribonuclease III/metabolism , Ribonuclease III/ultrastructureABSTRACT
A major challenge in human genetics is to identify the molecular mechanisms of trait-associated and disease-associated variants. To achieve this, quantitative trait locus (QTL) mapping of genetic variants with intermediate molecular phenotypes such as gene expression and splicing have been widely adopted1,2. However, despite successes, the molecular basis for a considerable fraction of trait-associated and disease-associated variants remains unclear3,4. Here we show that ADAR-mediated adenosine-to-inosine RNA editing, a post-transcriptional event vital for suppressing cellular double-stranded RNA (dsRNA)-mediated innate immune interferon responses5-11, is an important potential mechanism underlying genetic variants associated with common inflammatory diseases. We identified and characterized 30,319 cis-RNA editing QTLs (edQTLs) across 49 human tissues. These edQTLs were significantly enriched in genome-wide association study signals for autoimmune and immune-mediated diseases. Colocalization analysis of edQTLs with disease risk loci further pinpointed key, putatively immunogenic dsRNAs formed by expected inverted repeat Alu elements as well as unexpected, highly over-represented cis-natural antisense transcripts. Furthermore, inflammatory disease risk variants, in aggregate, were associated with reduced editing of nearby dsRNAs and induced interferon responses in inflammatory diseases. This unique directional effect agrees with the established mechanism that lack of RNA editing by ADAR1 leads to the specific activation of the dsRNA sensor MDA5 and subsequent interferon responses and inflammation7-9. Our findings implicate cellular dsRNA editing and sensing as a previously underappreciated mechanism of common inflammatory diseases.
Subject(s)
Adenosine Deaminase , Genetic Predisposition to Disease , Immune System Diseases , Inflammation , RNA Editing , RNA, Double-Stranded , Adenosine/metabolism , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Alu Elements/genetics , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Genome-Wide Association Study , Humans , Immune System Diseases/genetics , Immune System Diseases/immunology , Immune System Diseases/pathology , Immunity, Innate , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Inosine/metabolism , Interferon-Induced Helicase, IFIH1/metabolism , Interferons/genetics , Interferons/immunology , Quantitative Trait Loci/genetics , RNA Editing/genetics , RNA, Double-Stranded/genetics , RNA-Binding Proteins/metabolismABSTRACT
The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) modification in zebrafish early embryos, we found that m5C-modified maternal mRNAs display higher stability than non-m5C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m5C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1's cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m5C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m5C mRNA modification in early development.
Subject(s)
5-Methylcytosine/metabolism , Embryo, Nonmammalian/embryology , Embryonic Development/physiology , RNA Stability/physiology , RNA, Messenger, Stored/metabolism , Zebrafish/embryology , Animals , HeLa Cells , Humans , Mice , RNA, Messenger, Stored/genetics , Zebrafish/geneticsABSTRACT
Riboswitches are conserved regulatory RNA elements participating in various metabolic pathways. Recently, a novel RNA motif known as the folE RNA motif was discovered upstream of folE genes. It specifically senses tetrahydrofolate (THF) and is therefore termed THF-II riboswitch. To unravel the ligand recognition mechanism of this newly discovered riboswitch and decipher the underlying principles governing its tertiary folding, we determined both the free-form and bound-form THF-II riboswitch in the wild-type sequences. Combining structural information and isothermal titration calorimetry (ITC) binding assays on structure-based mutants, we successfully elucidated the significant long-range interactions governing the function of THF-II riboswitch and identified additional compounds, including alternative natural metabolites and potential lead compounds for drug discovery, that interact with THF-II riboswitch. Our structural research on the ligand recognition mechanism of the THF-II riboswitch not only paves the way for identification of compounds targeting riboswitches, but also facilitates the exploration of THF analogs in diverse biological contexts or for therapeutic applications.
Subject(s)
Nucleic Acid Conformation , Riboswitch , Tetrahydrofolates , Riboswitch/genetics , Tetrahydrofolates/chemistry , Tetrahydrofolates/metabolism , Ligands , Models, Molecular , RNA Folding , Nucleotide Motifs , MutationABSTRACT
African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. Although it has been extensively studied in the past, no vaccine or other useful treatment against ASFV is available. The genome of ASFV encodes more than 170 proteins, but the structures and functions for the majority of the proteins remain elusive, which hindered our understanding on the life cycle of ASFV and the development of ASFV-specific inhibitors. Here, we report the structural and biochemical studies of the highly conserved C962R protein of ASFV, showing that C962R is a multidomain protein. The N-terminal AEP domain is responsible for the DNA polymerization activity, whereas the DNA unwinding activity is catalyzed by the central SF3 helicase domain. The middle PriCT2 and D5_N domains and the C-terminal Tail domain all contribute to the DNA unwinding activity of C962R. C962R preferentially works on forked DNA, and likely functions in Base-excision repair (BER) or other repair pathway in ASFV. Although it is not essential for the replication of ASFV, C962R can serve as a model and provide mechanistic insight into the replicative primase proteins from many other species, such as nitratiruptor phage NrS-1, vaccinia virus (VACV) and other viruses.
Subject(s)
African Swine Fever Virus , Viral Proteins , Animals , African Swine Fever/virology , African Swine Fever Virus/enzymology , Swine , Viral Proteins/chemistry , Viral Proteins/metabolism , DNA Topoisomerases, Type I/chemistry , DNA ReplicationABSTRACT
[This corrects the article DOI: 10.1371/journal.pbio.2001272.].
ABSTRACT
MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.
Subject(s)
Antiporters/chemistry , Bacterial Proteins/chemistry , Ion Channel Gating/physiology , Antiporters/metabolism , Bacterial Proteins/metabolism , Binding Sites/drug effects , Biological Transport , Cryoelectron Microscopy , Crystallography, X-Ray , Cytoplasm/metabolism , Ion Channel Gating/drug effects , Kinetics , Magnesium/metabolism , Magnesium/pharmacology , Models, Molecular , Protein Domains/drug effects , Protein Domains/physiology , Protein Structure, Quaternary , Protein Structure, Secondary , Thermus thermophilus/metabolismABSTRACT
Neurospora crassa protein QDE-1, a member of the two-barrel polymerase superfamily, possesses both DNA- and RNA-dependent RNA polymerase (DdRP and RdRP) activities. The dual activities are essential for the production of double-stranded RNAs (dsRNAs), the precursors of small interfering RNAs (siRNAs) in N. crassa. Here, we report five complex structures of N-terminal truncated QDE-1 (QDE-1ΔN), representing four different reaction states: DNA/RNA-templated elongation, the de novo initiation of RNA synthesis, the first step of nucleotide condensation during de novo initiation and initial NTP loading. The template strand is aligned by a bridge-helix and double-psi beta-barrels 2 (DPBB2), the RNA product is held by DPBB1 and the slab domain. The DNA template unpairs with the RNA product at position -7, but the RNA template remains paired. The NTP analog coordinates with cations and is precisely positioned at the addition site by a rigid trigger loop and a proline-containing loop in the active center. The unique C-terminal tail from the QDE-1 dimer partner inserts into the substrate-binding cleft and plays regulatory roles in RNA synthesis. Collectively, this work elucidates the conserved mechanisms for DNA/RNA-dependent dual activities by QDE-1 and other two-barrel polymerase superfamily members.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Neurospora crassa/metabolism , RNA-Dependent RNA Polymerase/metabolism , Neurospora crassa/chemistry , Nucleotides , RNA, Double-Stranded , RNA, Small Interfering/metabolismABSTRACT
Wheat stripe rust is a destructive disease worldwide, caused by Puccinia striiformis f. sp. tritici (Pst). Resistance breeding is the most effective method of controlling stripe rust. Xinjiang is a relatively independent epidemic region of wheat stripe rust in China. In recent years, wheat stripe rust in this area has shown an upward trend. Therefore, the purpose of this study was to evaluate the resistance level of wheat cultivars (lines) to the prevalent Pst races and determine the genetic background of stripe rust resistance genes in Xinjiang. Six predominant Pst races in China were used to study resistance of 286 wheat cultivars (lines) at both seedling under controlled conditions and adult-plant stages under field conditions. In the seedling tests, 175 (61.19%) entries were resistant to races CYR23, 125 (43.71%) to CYR29, 153 (53.50%) to CYR31, 88 (30.77%) to CYR32, 174 (60.84%) to CYR33, and 98 (34.27%) to CYR34. Among the resistant entries, 23 (8.04%) were resistant to all six races. In the field test, 135 (47.20%) entries were resistant to the tested mixed races. Through comparing the responses in the seedling and adult-plant stages, 109 (38.11%) entries were found to have adult-plant resistance (APR), and 14 (4.90%) entries have all-stage resistance (ASR). The 286 wheat entries were also tested using a wheat breeder chip containing 12 Yr resistance loci. Among these entries, 44 (15.38%) were found to have single gene, 221 (77.27%) have two or more genes, and 21 (7.34%) have none of the 12 genes, including 144 (50.35%) with Yr30 and 5 (1.75%) with YrSP. Entries with two or more genes have stronger resistance to Pst. Overall, the majority of entries have all-stage and/or adult-plant resistance, but their genes for resistance in addition to the 12 tested Yr genes need to be determined. It is also necessary to introduce more effective resistance genes in the breeding programs to improve stripe rust resistance in wheat cultivars in Xinjiang.
ABSTRACT
Puccinia striiformis f. sp. tritici (Pst) is a destructive pathogen that causes wheat stripe rust worldwide. Understanding the population structure and dynamic of pathogen spread is critical to fight against this disease. Limited information is available for the population genetic structure of Pst in Uzbekistan, Central Asia. In this study, we carried out surveillance from 9 different regions (Andijan, Fergana, Jizzakh, Kashkadarya, Namangan, Samarkand, Sirdaryo, Surkhandarya and Tashkent) of Uzbekistan to fill this gap. A total of 255 isolates were collected, which were genotyped using 17 polymorphic simple sequence repeats (SSR) markers. The DAPC analysis results showed no population subdivision in these sample-collected regions except Surkhandarya. Multilocus genotype (MLG) analysis, FST, and Nei's genetic distance results indicated a clonal population (rBarD ≤ 0.12) and merely three MLGs accounting for 70% of the overall population. MLG-34 was predominant in all Uzbekistan regions, followed by MLG-36 and MLG-42. Low genotypic diversity was observed in Andijan, Fergana, Jizzakh, Kashkadarya, Namangan, Sirdaryo, and Tashkent (0.56 to 0.76), compared with Samarkand (0.82) and Surkhandarya (0.97). No virulence against Yr5, Yr15, YrSp, and Yr26 was found, while resistant was overcome against Yr1, Yr2, Yr6, Yr9, Yr17, and Yr44 genes (Virulence frequency =≥75%). Comparative study results of Uzbekistan with previous Himalayan population were showed divergence from China and Pakistan populations. Further studies need to be conducted in a worldwide context to understand migration patterns; for that purpose, collaborative work is essential due to the Pst long-distance migration capability.
ABSTRACT
The regulation of intracellular pH in yeast (Saccharomyces cerevisiae) cells is critical for cell function and viability. In yeast, protons (H+) can be excreted from the cell by plasma membrane ATPase PMA1 and pumped into vacuoles by vacuolar H+-ATPase. Because PMA1 is critical to the survival of yeast cells, it is unknown whether other compensatory components are involved in pH homeostasis in the absence of PMA1. To elucidate how intracellular pH is regulated independently of PMA1, we employed a screening approach by exposing the yeast haploid deletion mutant library (ver 4.0) to the selective plant plasma membrane H+-ATPase inhibitor PS-1, which we previously reported. After repeated screenings and verification, we identified two proteins, Aly1 and Aly2, that play a role in the regulation of intracellular pH when PMA1 is deficient. Our research uncovers a new perspective on the regulation of intracellular pH related to PMA1 and also preliminarily reveals a role for Aly1 and Aly2 in the regulation of intracellular pH.
ABSTRACT
A Gram-stain-negative, strictly aerobic, motile, slightly curved rod-shaped bacterium with multiple flagella, designated strain EGI 63088T, was isolated from a bulk soil of Kalidium foliatum, collected from Wujiaqu in Xinjiang Uighur Autonomous Region, PR China. The optimal growth temperature, salinity, and pH for strain EGI 63088T growth were 30 °C, 3% (w/v) NaCl and 8, respectively. Phylogenetic analysis using 16S rRNA gene sequences indicated that strain EGI 63088T showed the highest sequence similarities to Halomonas heilongjiangensis 9-2T (97.94%), H. lysinitropha 3(2)T (97.51%), and H. daqiaonensis CGMCC 1.9150T (97.08%). The average nucleotide identity and digital DNA-DNA hybridization values between the strain EGI 63088T and H. heilongjiangensis 9-2T were 89.03 and 41.10%, respectively. The DNA G + C content of the genome for strain EGI 63088T was 66.3 mol%. The most prevalent antibiotic resistance and virulence-related genes in Halomonas genomes were Streptomyces cinnamoneu EF-Tu mutant, pilT, and cheY, respectively. The predominant fatty acids of strain EGI 63088T were summed feature 8 (C18: 1 ω6c and/or C18: 1 ω7c), summed feature 3 (C16: 1 ω6c and/or C16: 1 ω7c), and C16: 0; its major respiratory quinone was ubiquinone-9 (Q-9), and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. According to the above results, strain EGI 63088T is considered a novel species of the genus Halomonas, for which the name Halomonas flagellata sp. nov. is proposed. The type strain is EGI 63088T (= KCTC 92047T = CGMCC 1.19133T).
Subject(s)
Halomonas , Halomonas/genetics , Phylogeny , RNA, Ribosomal, 16S/genetics , Sodium Chloride , CardiolipinsABSTRACT
Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs.
Subject(s)
Protozoan Proteins/chemistry , RNA Stability/physiology , RNA, Guide, Kinetoplastida/metabolism , RNA, Protozoan/metabolism , Ribonuclease III/chemistry , Trypanosoma brucei brucei/enzymology , Amino Acid Sequence , Base Sequence , Crystallography, X-Ray , Gene Expression Regulation , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , Protein Domains , Protozoan Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribonuclease III/metabolism , Structure-Activity Relationship , Substrate Specificity , Zinc FingersABSTRACT
Nucleosome Assembly Protein 1 (NAP1) family proteins are evolutionarily conserved histone chaperones that play important roles in diverse biological processes. In this study, we determined the crystal structure of Arabidopsis NAP1-Related Protein 1 (NRP1) complexed with H2A-H2B and uncovered a previously unknown interaction mechanism in histone chaperoning. Both in vitro binding and in vivo plant rescue assays proved that interaction mediated by the N-terminal α-helix (αN) domain is essential for NRP1 function. In addition, the C-terminal acidic domain (CTAD) of NRP1 binds to H2A-H2B through a conserved mode similar to other histone chaperones. We further extended previous knowledge of the NAP1-conserved earmuff domain by mapping the amino acids of NRP1 involved in association with H2A-H2B. Finally, we showed that H2A-H2B interactions mediated by αN, earmuff, and CTAD domains are all required for the effective chaperone activity of NRP1. Collectively, our results reveal multiple interaction modes of a NAP1 family histone chaperone and shed light on how histone chaperones shield H2A-H2B from nonspecific interaction with DNA.
Subject(s)
Histones/chemistry , Models, Molecular , Nucleosome Assembly Protein 1/chemistry , Amino Acid Motifs , Amino Acids , Arabidopsis , Binding Sites , Conserved Sequence , Crystallography, X-Ray , Histones/metabolism , Nucleosome Assembly Protein 1/metabolism , Protein Binding , Protein Conformation , Protein Interaction Domains and MotifsABSTRACT
Plant aquaporins are a recently noted biological resource with a great potential to improve crop growth and defense traits. Here, we report the functional modulation of the rice (Oryza sativa) aquaporin OsPIP1;3 to enhance rice photosynthesis and grain production and to control bacterial blight and leaf streak, the most devastating worldwide bacterial diseases in the crop. We characterize OsPIP1;3 as a physiologically relevant CO2 -transporting facilitator, which supports 30% of rice photosynthesis on average. This role is nullified by interaction of OsPIP1;3 with the bacterial protein Hpa1, an essential component of the Type III translocon that supports translocation of the bacterial Type III effectors PthXo1 and TALi into rice cells to induce leaf blight and streak, respectively. Hpa1 binding shifts OsPIP1;3 from CO2 transport to effector translocation, aggravates bacterial virulence, and blocks rice photosynthesis. On the contrary, the external application of isolated Hpa1 to rice plants effectively prevents OsPIP1;3 from interaction with Hpa1 secreted by the bacteria that are infecting the plants. Blockage of the OsPIP1;3-Hpa1 interaction reverts OsPIP1;3 from effector translocation to CO2 transport, abrogates bacterial virulence, and meanwhile induces defense responses in rice. These beneficial effects can combine to enhance photosynthesis by 29-30%, reduce bacterial disease by 58-75%, and increase grain yield by 11-34% in different rice varieties investigated in small-scale field trials conducted during the past years. Our results suggest that crop productivity and immunity can be coordinated by modulating the physiological and pathological functions of a single aquaporin to break the growth-defense tradeoff barrier.
Subject(s)
Oryza/physiology , Photosynthesis/physiology , Plant Proteins/metabolism , Xanthomonas/pathogenicity , Bacterial Proteins/metabolism , Biological Transport , Carbon Dioxide/metabolism , China , Gene Expression Regulation, Plant , Host-Pathogen Interactions/physiology , Oryza/microbiology , Plant Diseases/microbiology , Plant Leaves/physiology , Plant Proteins/genetics , Plants, Genetically Modified , Seeds/genetics , Seeds/growth & development , Virulence , Xanthomonas/metabolismABSTRACT
The basic helix-loop-helix (bHLH) family is one of the most conserved transcription factor families that plays an important role in regulating cell growth, differentiation and tissue development. Typically, members of this family form homo- or heterodimers to recognize specific motifs and activate transcription. MyoD is a vital transcription factor that regulates muscle cell differentiation. However, it is necessary for MyoD to form a heterodimer with E-proteins to activate transcription. Even though the crystal structure of the MyoD homodimer has been determined, the structure of the MyoD heterodimer in complex with the E-box protein remains unclear. In this study, we determined the crystal structure of the bHLH domain of the MyoD-E47 heterodimer at 2.05 Å. Our structural analysis revealed that MyoD interacts with E47 through a hydrophobic interface. Moreover, we confirmed that heterodimerization could enhance the binding affinity of MyoD to E-box sequences. Our results provide new structural insights into the heterodimer of MyoD and E-box protein, suggesting the molecular mechanism of transcription activation of MyoD upon binding to E-box protein.
Subject(s)
DNA-Binding Proteins , MyoD Protein , DNA-Binding Proteins/metabolism , Helix-Loop-Helix Motifs , MyoD Protein/metabolism , Protein Binding , TCF Transcription Factors/metabolism , Transcription Factor 7-Like 1 Protein/metabolism , Transcription Factors/metabolismABSTRACT
Tuberculosis (TB), caused by infection with airborne Mycobacterium tuberculosis (MTB), seriously threatens human health and has become a public health problem of worldwide concern. To achieve effective control of TB, rapid and sensitive detection of MTB is particularly important. At present, the common detection methods for MTB cannot meet the requirements of speed, flexibility and portability simultaneously. In this work, a multichannel microfluidic chip was developed and packaged with an ultra-sensitive silicon nanowire field-effect-transistor biosensor. The fluid system was tested and optimized through simulation, and the best conditions were determined: the flow rate was 0.3 mL min-1 and the flow direction was perpendicular to a silicon nanowire. A one-way valve, a switching valve and a peristaltic pump were combined to establish a biosensor detection system to realize the automatic detection of TB samples. Then we systematically explained the factors affecting simulated exhaled breath condensate (SEBC) collection, and established and optimized the method for collection of SEBC from the perspective of collection volume and biological activity. The best collection conditions were determined for a 5 mm pipe diameter at 0 °C, and a sufficient sample volume was obtained in only 2 minutes for microfluidic detection. Then, the actual application value of the established collection method was further evaluated. Volunteers were recruited and this method was used to collect their exhaled breath condensate to analyze the collection effect. The system detected MTB in SEBC with good sensitivity (â¼4 × 104 particles per mL). It is expected to be further integrated and miniaturized in the future to realize point-of-care testing.
Subject(s)
Biosensing Techniques , Mycobacterium tuberculosis , Tuberculosis , Bacterial Proteins , Humans , Point-of-Care Testing , Sensitivity and SpecificityABSTRACT
We have solved two families of crystal structures of the human Dicer "platform-PAZ-connector helix" cassette in complex with small interfering RNAs (siRNAs). The structures possess two adjacently positioned pockets: a 2 nt 3'-overhang-binding pocket within the PAZ domain (3' pocket) and a phosphate-binding pocket within the platform domain (phosphate pocket). One family of complexes contains a knob-like α-helical protrusion, designated "hDicer-specific helix," that separates the two pockets and orients the bound siRNA away from the surface of Dicer, which could be indicative of a product release/transfer state. In the second complex, the helical protrusion is melted/disordered and the bound siRNA is aligned toward the surface of Dicer, suggestive of a cleavage-competent state. These structures allow us to propose that the transition from the cleavage-competent to the postulated product release/transfer state may involve release of the 5'-phosphate from the phosphate pocket while retaining the 3' overhang in the 3' pocket.
Subject(s)
DEAD-box RNA Helicases/chemistry , Ribonuclease III/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Crystallography, X-Ray , DEAD-box RNA Helicases/metabolism , Humans , Mice , Mice, Knockout , Molecular Sequence Data , Mutation , Phosphates/chemistry , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Small Interfering/metabolism , Ribonuclease III/metabolism , Sequence Homology, Amino Acid , Surface Plasmon ResonanceABSTRACT
NrS-1 is the first known phage that can infect Epsilonproteobacteria, one of the predominant primary producers in the deep-sea hydrothermal vent ecosystems. NrS-1 polymerase is a multidomain enzyme and is one key component of the phage replisome. The N-terminal Prim/Pol and HBD domains are responsible for DNA polymerization and de novo primer synthesis activities of NrS-1 polymerase. However, the structure and function of the C-terminus (CTR) of NrS-1 polymerase are poorly understood. Here, we report two crystal structures, showing that NrS-1 CTR adopts one unique hexameric ring-shaped conformation. Although the central helicase domain of NrS-1 CTR shares structural similarity with the superfamily III helicases, the folds of the Head and Tail domains are completely novel. Via mutagenesis and in vitro biochemical analysis, we identified many residues important for the helicase and polymerization activities of NrS-1 polymerase. In addition to NrS-1 polymerase, our study may also help us identify and understand the functions of multidomain polymerases expressed by many NrS-1 related phages.
Subject(s)
Bacteriophages/enzymology , DNA Replication/genetics , DNA-Directed DNA Polymerase/ultrastructure , Protein Conformation , Amino Acid Sequence/genetics , Bacteriophages/genetics , Bacteriophages/ultrastructure , Crystallography, X-Ray , DNA-Directed DNA Polymerase/chemistry , Ecosystem , Epsilonproteobacteria/genetics , Epsilonproteobacteria/virology , Hydrothermal Vents/chemistryABSTRACT
Phytophthora are eukaryotic pathogens that cause enormous losses in agriculture and forestry. Each Phytophthora species encodes hundreds of effector proteins that collectively have essential roles in manipulating host cellular processes and facilitating disease development. Here we report the crystal structure of the effector Phytophthora suppressor of RNA silencing 2 (PSR2). PSR2 produced by the soybean pathogen Phytophthora sojae (PsPSR2) consists of seven tandem repeat units, including one W-Y motif and six L-W-Y motifs. Each L-W-Y motif forms a highly conserved fold consisting of five α-helices. Adjacent units are connected through stable, directional linkages between an internal loop at the C terminus of one unit and a hydrophobic pocket at the N terminus of the following unit. This unique concatenation results in an overall stick-like structure of PsPSR2. Genome-wide analyses reveal 293 effectors from five Phytophthora species that have the PsPSR2-like arrangement, that is, containing a W-Y motif as the "start" unit, various numbers of L-W-Y motifs as the "middle" units, and a degenerate L-W-Y as the "end" unit. Residues involved in the interunit interactions show significant conservation, suggesting that these effectors also use the conserved concatenation mechanism. Furthermore, functional analysis demonstrates differential contributions of individual units to the virulence activity of PsPSR2. These findings suggest that the L-W-Y fold is a basic structural and functional module that may serve as a "building block" to accelerate effector evolution in Phytophthora.