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a nondiscriminating glutamyl-trna synthetase in the plasmodium apicoplast: the first enzyme in an indirect aminoacylation pathway.the malaria parasite plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. we identified components of an indirect aminoacylation pathway for gln-trna(gln) biosynthesis in plasmodium that we hypothesized would be essential for api ...201324072705
mutations in gtp binding protein obg of mycoplasma synoviae vaccine strain ms-h: implications in temperature-sensitivity phenotype.mycoplasma synoviae strain ms-h, developed by chemical mutagenesis of the australian field strain 86079/7ns, is a live temperature-sensitive (ts (+)) vaccine used for control of m. synoviae infection in poultry worldwide. genetic basis of temperature sensitivity and attenuation of ms-h has not been revealed thus far. comparison of the complete genome sequence of ms-h, its parent strain 86079/7ns and two non-temperature sensitive (ts (-)) reisolates of ms-h revealed a mutation in a highly conserv ...201324069254
initiation of mrna decay in bacteria.the instability of messenger rna is fundamental to the control of gene expression. in bacteria, mrna degradation generally follows an "all-or-none" pattern. this implies that if control is to be efficient, it must occur at the initiating (and presumably rate-limiting) step of the degradation process. studies of e. coli and b. subtilis, species separated by 3 billion years of evolution, have revealed the principal and very disparate enzymes involved in this process in the two organisms. the early ...201324064983
initiation of mrna decay in bacteria.the instability of messenger rna is fundamental to the control of gene expression. in bacteria, mrna degradation generally follows an "all-or-none" pattern. this implies that if control is to be efficient, it must occur at the initiating (and presumably rate-limiting) step of the degradation process. studies of e. coli and b. subtilis, species separated by 3 billion years of evolution, have revealed the principal and very disparate enzymes involved in this process in the two organisms. the early ...201324064983
functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter.targeting sequencing to genes involved in key environmental processes, i.e., ecofunctional genes, provides an opportunity to sample nature's gene guilds to greater depth and help link community structure to process-level outcomes. vastly different approaches have been implemented for sequence processing and, ultimately, for taxonomic placement of these gene reads. the overall quality of next generation sequence analysis of functional genes is dependent on multiple steps and assumptions of unknow ...201324062736
molecular mechanism and physiological role of active-deactive transition of mitochondrial complex i.the unique feature of mitochondrial complex i is the so-called a/d transition (active-deactive transition). the a-form catalyses rapid oxidation of nadh by ubiquinone (k ~104 min-1) and spontaneously converts into the d-form if the enzyme is idle at physiological temperatures. such deactivation occurs in vitro in the absence of substrates or in vivo during ischaemia, when the ubiquinone pool is reduced. the d-form can undergo reactivation given both nadh and ubiquinone availability during slow ( ...201324059527
an ftsh protease is recruited to the mitochondrion of plasmodium falciparum.the two organelles, apicoplast and mitochondrion, of the malaria parasite plasmodium falciparum have unique morphology in liver and blood stages; they undergo complex branching and looping prior to division and segregation into daughter merozoites. little is known about the molecular processes and proteins involved in organelle biogenesis in the parasite. we report the identification of an aaa+/ftsh protease homolog (pfftsh1) that exhibits atp- and zn(2+)-dependent protease activity. pfftsh1 und ...201324058559
loose binding of the df axis with the a3b3 complex stimulates the initial activity of enterococcus hirae v1-atpase.vacuolar atpases (v-atpases) function as proton pumps in various cellular membrane systems. the hydrophilic v1 portion of the v-atpase is a rotary motor, in which a central-axis df complex rotates inside a hexagonally arranged catalytic a3b3 complex by using atp hydrolysis energy. we have previously reported crystal structures of enterococcushirae v-atpase a3b3 and a3b3df (v1) complexes; the result suggested that the df axis induces structural changes in the a3b3 complex through extensive protei ...201324058539
membrane chaperone secdf plays a role in the secretion of listeria monocytogenes major virulence factors.listeria monocytogenes is a gram-positive human intracellular pathogen that infects diverse mammalian cells. upon invasion, l. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. it has been presumed, but was not empirically established, that the sec translocation system is the primary mediator of this secretion. here, we validate an important role for secdf, a component of the sec system, in the secretion of several critical l. monocytoge ...201324056100
structure of the mtb card/rnap β-lobes complex reveals the molecular basis of interaction and presents a distinct dna-binding domain for mtb card.card from mycobacterium tuberculosis (mtb) is an essential protein shown to be involved in stringent response through downregulation of rrna and ribosomal protein genes. card interacts with the β-subunit of rnap and this interaction is vital for mtb's survival during the persistent infection state. we have determined the crystal structure of card in complex with the rnap β-subunit β1 and β2 domains at 2.1 å resolution. the structure reveals the molecular basis of card/rnap interaction, providing ...201324055315
tools for characterizing bacterial protein synthesis inhibitors.many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. however, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. i ...201324041905
systematic mutational analysis of the putative hydrolase pqse: toward a deeper molecular understanding of virulence acquisition in pseudomonas aeruginosa.pseudomonas aeruginosa is an important opportunistic human pathogen that can establish bacterial communication by synchronizing the behavior of individual cells in a molecular phenomenon known as "quorum sensing". through an elusive mechanism involving gene products of the pqs operon, the pqse enzyme is absolutely required for the synthesis of extracellular phenazines, including the toxic blue pigment pyocyanin, effectively allowing cells to achieve full-fledged virulence. despite several functi ...201324040042
effects of upconversion nanoparticles on polymerase chain reaction.nanoparticles (nps) are attractive materials owing to their physical and electrochemical properties, which make them extremely useful in diagnostic applications. photon upconversion is the phenomenon where high-energy photons are emitted upon excitation of low-energy photons. nucleic acids detection based on upconversion nanoparticles (ucnps), which display a high signal-to-noise ratio and no photobleaching, has been widely applied. we evaluated whether ucnps can improve polymerase chain reactio ...201324039935
complete genomic dna sequence of the east asian spotted fever disease agent rickettsia japonica.rickettsia japonica is an obligate intracellular alphaproteobacteria that causes tick-borne japanese spotted fever, which has spread throughout east asia. we determined the complete genomic dna sequence of r. japonica type strain yh (vr-1363), which consists of 1,283,087 base pairs (bp) and 971 protein-coding genes. comparison of the genomic dna sequence of r. japonica with other rickettsiae in the public databases showed that 2 regions (4,323 and 216 bp) were conserved in a very narrow range of ...201324039725
the uve1 endonuclease is regulated by the white collar complex to protect cryptococcus neoformans from uv damage.the pathogenic fungus cryptococcus neoformans uses the bwc1-bwc2 photoreceptor complex to regulate mating in response to light, virulence and ultraviolet radiation tolerance. how the complex controls these functions is unclear. here, we identify and characterize a gene in cryptococcus, uve1, whose mutation leads to a uv hypersensitive phenotype. the homologous gene in fission yeast schizosaccharomyces pombe encodes an apurinic/apyrimidinic endonuclease acting in the uvde-dependent excision repai ...201324039606
phosphorylation and acetylation of acyl-coa synthetase- i.long chain acyl-coa synthetase 1 (acsl1) contributes 50 to 90% of total acsl activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. although the functional importance of acsl1 is becoming clear, little is understood about its post-translational regulation. in order to investigate the post-translational modifications of acsl1 under different physiological conditions, we overexpressed acsl1 in hepatocytes, brown adipocytes, and 3t3-l1 diffe ...201124039348
bacterial argonaute samples the transcriptome to identify foreign dna.eukaryotic argonautes bind small rnas and use them as guides to find complementary rna targets and induce gene silencing. though homologs of eukaryotic argonautes are present in many bacteria and archaea, their small rna partners and functions are unknown. we found that the argonaute of rhodobacter sphaeroides (rsago) associates with 15-19 nt rnas that correspond to the majority of transcripts. rsago also binds single-stranded 22-24 nt dna molecules that are complementary to the small rnas and e ...201324034694
glucose(xylose) isomerase production by streptomyces sp. ch7 grown on agricultural residues.streptomyces sp. ch7 was found to efficiently produce glucose(xylose) isomerase when grown on either xylan or agricultural residues. this strain produced a glucose(xylose) isomerase activity of roughly 1.8 u/mg of protein when it was grown in medium containing 1% xylose as a carbon source. maximal enzymatic activities of about 5 and 3 u/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. the enzyme was purified from a mycelial extract to 16-fold purity with only two conse ...201224031932
evaluation of biomass production, carotenoid level and antioxidant capacity produced by thermus filiformis using fractional factorial design.a fractional factorial design 2(5-1) was used to evaluate the effect of temperature, ph, and concentrations of yeast extract, tryptone and nitsch's trace elements on the biomass, total carotenoids and protection against singlet oxygen by carotenoid extracts of the bacterium thermus filiformis. in addition, the carotenoid composition was determined by high-performance liquid chromatography connected to a diode array and mass spectrometer detectors (hplc-dad-ms/ms). the production of biomass range ...201224031811
initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5b and mechanism among translational gtpases.the initiation of protein synthesis uses initiation factor 2 (if2) in prokaryotes and a related protein named eukaryotic initiation factor 5b (eif5b) in eukaryotes. if2 is a gtpase that positions the initiator trna on the 30s ribosomal initiation complex and stimulates its assembly to the 50s ribosomal subunit to make the 70s ribosome. the 3.1-å resolution x-ray crystal structures of the full-length thermus thermophilus apo if2 and its complex with gdp presented here exhibit two different confor ...201324029018
involvement of protein if2 n domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor.translation initiation factor 2 (if2) promotes 30s initiation complex (ic) formation and 50s subunit joining, which produces the 70s ic. the architecture of full-length if2, determined by small angle x-ray diffraction and cryo electron microscopy, reveals a more extended conformation of if2 in solution and on the ribosome than in the crystal. the n-terminal domain is only partially visible in the 30s ic, but in the 70s ic, it stabilizes interactions between if2 and the l7/l12 stalk of the 50s, a ...201324029017
chaperone machines for protein folding, unfolding and disaggregation.molecular chaperones are diverse families of multidomain proteins that have evolved to assist nascent proteins to reach their native fold, protect subunits from heat shock during the assembly of complexes, prevent protein aggregation or mediate targeted unfolding and disassembly. their increased expression in response to stress is a key factor in the health of the cell and longevity of an organism. unlike enzymes with their precise and finely tuned active sites, chaperones are heavy-duty molecul ...201324026055
splicefinder - a fast and easy screening method for active protein trans-splicing positions.split intein enabled protein trans-splicing (pts) is a powerful method for the ligation of two protein fragments, thereby paving the way for various protein modification or protein function control applications. pts activity is strongly influenced by the amino acids directly flanking the splice junctions. however, to date no reliable prediction can be made whether or not a split intein is active in a particular foreign extein context. here we describe splicefinder, a pcr-based method, allowing f ...201324023792
exploration of freely available web-interfaces for comparative homology modelling of microbial proteins.this study was conducted to find the best suited freely available software for modelling of proteins by taking a few sample proteins. the proteins used were small to big in size with available crystal structures for the purpose of benchmarking. key players like phyre2, swiss-model, cphmodels-3.0, homer, (ps)2, (ps)(2)-v(2), modweb were used for the comparison and model generation.201324023424
aminoacylation of trna 2'- or 3'-hydroxyl by phosphoseryl- and pyrrolysyl-trna synthetases.class i and ii aminoacyl-trna synthetases (aarss) attach amino acids to the 2'- and 3'-oh of the trna terminal adenosine, respectively. one exception is phenylalanyl-trna synthetase (phers), which belongs to class ii but attaches phenylalanine to the 2'-oh. here we show that two class ii aarss, o-phosphoseryl- (seprs) and pyrrolysyl-trna (pylrs) synthetases, aminoacylate the 2'- and 3'-oh, respectively. structure-based-phylogenetic analysis reveals that seprs is more closely related to phers tha ...201324021645
thermus oshimai jl-2 and t. thermophilus jl-18 genome analysis illuminates pathways for carbon, nitrogen, and sulfur cycling.the complete genomes of thermus oshimai jl-2 and t. thermophilus jl-18 each consist of a circular chromosome, 2.07 mb and 1.9 mb, respectively, and two plasmids ranging from 0.27 mb to 57.2 kb. comparison of the t. thermophilus jl-18 chromosome with those from other strains of t. thermophilus revealed a high degree of synteny, whereas the megaplasmids from the same strains were highly plastic. the t. oshimai jl-2 chromosome and megaplasmids shared little or no synteny with other sequenced thermu ...201324019992
the nitric-oxide reductase from paracoccus denitrificans uses a single specific proton pathway.the no reductase from paracoccus denitrificans reduces no to n2o (2no + 2h(+) + 2e(-) → n2o + h2o) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent no reductase; cnor). cnors are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the o2-reducing, proton-pumping respiratory enzymes. in contrast, although no reduction is as exergonic as o2 reduction, there are no protons pumped in cnor, and in addition, protons needed for no reduction ...201324014024
the tetr family of regulators.the most common prokaryotic signal transduction mechanisms are the one-component systems in which a single polypeptide contains both a sensory domain and a dna-binding domain. among the >20 classes of one-component systems, the tetr family of regulators (tfrs) are widely associated with antibiotic resistance and the regulation of genes encoding small-molecule exporters. however, tfrs play a much broader role, controlling genes involved in metabolism, antibiotic production, quorum sensing, and ma ...201324006471
type iv pili in gram-positive bacteria.type iv pili (t4p) are surface-exposed fibers that mediate many functions in bacteria, including locomotion, adherence to host cells, dna uptake (competence), and protein secretion and that can act as nanowires carrying electric current. t4p are composed of a polymerized protein, pilin, and their assembly apparatuses share protein homologs with type ii secretion systems in eubacteria and the flagella of archaea. t4p are found throughout gram-negative bacterial families and have been studied most ...201324006467
ppr-smrs: ancient proteins with enigmatic functions.a small subset of the large pentatricopeptide repeat (ppr) protein family in higher plants contain a c-terminal small muts-related (smr) domain. although few in number, they figure prominently in the chloroplast biogenesis and retrograde signaling literature due to their striking mutant phenotypes. in this review, we summarize current knowledge of ppr-smr proteins focusing on arabidopsis and maize proteomic and mutant studies. we also examine their occurrence in other organisms and have determin ...201324004908
single mutations that redirect internal proton transfer in the ba3 oxidase from thermus thermophilus.the ba3-type cytochrome c oxidase from thermus thermophilus is a membrane-bound proton pump. results from earlier studies have shown that with the aa3-type oxidases proton uptake to the catalytic site and "pump site" occurs simultaneously. however, with ba3 oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than that with the aa3 oxidases. in addition, the timing of formation and decay of catalytic intermediates is dif ...201324004023
argonaute protein as a linker to command center of physiological processes.micrornas (mirnas) post-transcriptionally regulate gene expression by binding to target mrnas with perfect or imperfect complementarity, recruiting an argonaute (ago) protein complex that usually results in degradation or translational repression of the target mrna. ago proteins function as the slicer enzyme in mirna and small interfering rna (sirna) pathways involved in human physiological and pathophysiological processes, such as antiviral responses and disease formation. although the past dec ...201323997530
exploration of deinococcus-thermus molecular diversity by novel group-specific pcr primers.the deeply branching deinococcus-thermus lineage is recognized as one of the most extremophilic phylum of bacteria. in previous studies, the presence of deinococcus-related bacteria in the hot arid tunisian desert of tataouine was demonstrated through combined molecular and culture-based approaches. similarly, thermus-related bacteria have been detected in tunisian geothermal springs. the present work was conducted to explore the molecular diversity within the deinococcus-thermus phylum in these ...201323996915
a two-domain elevator mechanism for sodium/proton antiport.sodium/proton (na(+)/h(+)) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. in humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. the best understood model system for na(+)/h(+) antiport is nhaa from escherichia coli, for which both electron microscopy and crystal structures are available. nhaa is made up of two distinct domains: a core domain and a dimeriza ...201323995679
divergent contributions of conserved active site residues to transcription by eukaryotic rna polymerases i and ii.multisubunit rna polymerases (msrnaps) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msrnap functional conservation. however, we show that mutations in the trigger loop (tl) in the largest subunit of rna polymerase i (pol i) yield phenotypes unexpected from studies of pol ii. for example, a well-characterized gain-of-function mutation in pol ii results in loss of function in pol i (pol ii: rpb1- e1103g; pol i: rpa190- ...201323994471
structural phylogenomics retrodicts the origin of the genetic code and uncovers the evolutionary impact of protein flexibility.the genetic code shapes the genetic repository. its origin has puzzled molecular scientists for over half a century and remains a long-standing mystery. here we show that the origin of the genetic code is tightly coupled to the history of aminoacyl-trna synthetase enzymes and their interactions with trna. a timeline of evolutionary appearance of protein domain families derived from a structural census in hundreds of genomes reveals the early emergence of the 'operational' rna code and the late i ...201323991065
generation of a mouse model with down-regulated u50 snorna (snord50) expression and its organ-specific phenotypic modulation.box c/d-type small nucleolar rnas (snornas) are functional rnas responsible for mediating 2'-o-ribose methylation of ribosomal rnas (rrnas) within the nucleolus. in the past years, evidence for the involvement of human u50 snorna in tumorigenesis has been accumulating. we previously identified u50hg, a non-protein-coding gene that hosted a box c/d-type u50 snorna, in a chromosomal breakpoint in a human b-cell lymphoma. mouse genome analysis revealed four mouse u50 (mu50) host-genes: three mu50hg ...201323991050
serial femtosecond x-ray diffraction of 30s ribosomal subunit microcrystals in liquid suspension at ambient temperature using an x-ray free-electron laser.high-resolution ribosome structures determined by x-ray crystallography have provided important insights into the mechanism of translation. such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. here, the application of serial femtosecond x-ray crystallography (sfx) using an x-ray free-electron laser (xfel) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30s ribosomal ...201323989164
identifying ligand-binding hot spots in proteins using brominated fragments.high-quality crystals of thermus thermophilus ef-tu in the gtp-bound conformation at 1.7-2.7 å resolution were used to test 18 small organic molecules, all brominated for confident identification in the anomalous difference maps. from this relatively small collection, it was possible to identify a small molecule bound in the functionally important trna cca-end binding pocket. the antibiotic ge2270 a is known to interact with the same pocket in ef-tu and to disrupt the association with trna. brom ...201323989163
computational simulation strategies for analysis of multisubunit rna polymerases. 201323987500
the temperature sensitivity of a mutation in the essential trna modification enzyme trna methyltransferase d (trmd).conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. resolving these possibilities is important for exploiting the ts mutation f ...201323986443
cloning and characterization of ef-tu and ef-ts from pseudomonas aeruginosa.we have cloned genes encoding elongation factors ef-tu and ef-ts from pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. sequence analysis indicated that p. aeruginosa ef-tu and ef-ts are 84% and 55% identical to e. coli counterparts, respectively. p. aeruginosa ef-tu was active when assayed in gdp exchange assays. kinetic parameters for the interaction of ef-tu with gdp in the absence of ef-ts were observed to be k m = 33 μm, k cat (obs) = 0.003 s(-1 ...201323984384
investigating the function of [2fe-2s] cluster n1a, the off-pathway cluster in complex i, by manipulating its reduction potential.nadh:quinone oxidoreductase (complex i) couples nadh oxidation and quinone reduction to proton translocation across an energy-transducing membrane. all complexes i contain a flavin to oxidize nadh, seven iron-sulfur clusters to transfer electrons from the flavin to quinone and an eighth cluster (n1a) on the opposite side of the flavin. the role of cluster n1a is unknown, but escherichia coli complex i has an unusually high-potential cluster n1a and its reduced flavin produces h2o2, not superoxid ...201323980528
s6:s18 ribosomal protein complex interacts with a structural motif present in its own mrna.prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. in many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal rna, but also to a motif localized within their own mrna, and thereby regulate expression of the operon. in this study, we computationally predicted an rna motif present in many bacterial phyla within the 5' untranslated region of operons encoding ribosomal proteins s6 and s18. we demonstrated that the s6:s ...201323980204
crystal structure of ruvc resolvase in complex with holliday junction substrate.the key intermediate in genetic recombination is the holliday junction (hj), a four-way dna structure. at the end of recombination, hjs are cleaved by specific nucleases called resolvases. in gram-negative bacteria, this cleavage is performed by ruvc, a dimeric endonuclease that belongs to the retroviral integrase superfamily. here, we report the first crystal structure of ruvc in complex with a synthetic hj solved at 3.75 å resolution. the junction in the complex is in an unfolded 2-fold symmet ...201323980027
reca protein recruits structural maintenance of chromosomes (smc)-like recn protein to dna double-strand breaks.escherichia coli recn is an smc (structural maintenance of chromosomes) family protein that is required for dna double-strand break (dsb) repair. previous studies show that gfp-recn forms nucleoid-associated foci in response to dna damage, but the mechanism by which recn is recruited to the nucleoid is unknown. here, we show that the assembly of gfp-recn foci on the nucleoid in response to dna damage involves a functional interaction between recn and reca. a novel reca allele identified in this ...201323974212
simultaneous use of muts and reca for suppression of nonspecific amplification during pcr.thermus thermophilus muts, a thermostable mismatch-recognizing protein, is utilized in pcr to suppress nonspecific amplification by preventing synthesis from mismatched primers. t. thermophilus reca also decreases nonspecific amplification by promoting proper hybridization between the primer and template. we observed that muts and reca function under the same reaction conditions and that muts and reca do not preclude each other. furthermore, there were some dna sequences for which only one of th ...201323970956
automated classification of rna 3d motifs and the rna 3d motif atlas.the analysis of atomic-resolution rna three-dimensional (3d) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-watson-crick base pairs. structurally similar loops define rna 3d motifs that are conserved in homologous rna molecules, but can also occur at nonhomologous sites in diverse rnas, and which often vary in sequence. to further our understanding of rna motif structure and sequence variability and to provide a useful resource for ...201323970545
axial interactions in the mixed-valent cua active site and role of the axial methionine in electron transfer.within cu-containing electron transfer active sites, the role of the axial ligand in type 1 sites is well defined, yet its role in the binuclear mixed-valent cua sites is less clear. recently, the mutation of the axial met to leu in a cua site engineered into azurin (cua az) was found to have a limited effect on e(0) relative to this mutation in blue copper (bc). detailed low-temperature absorption and magnetic circular dichroism, resonance raman, and electron paramagnetic resonance studies on c ...201323964128
incorporation of nucleoside probes opposite o⁶-methylguanine by sulfolobus solfataricus dna polymerase dpo4: importance of hydrogen bonding.o⁶-methylguanine (o⁶-meg) is a mutagenic dna lesion, arising from the action of methylating agents on guanine (g) in dna. dpo4, an archaeal low-fidelity y-family dna polymerase involved in translesion dna synthesis (tls), is a model for studying how human y-family polymerases bypass dna adducts. previous work showed that dpo4-mediated dttp incorporation is favored opposite o⁶-meg rather than opposite g. however, factors influencing the preference of dpo4 to incorporate dttp opposite o⁶-meg are n ...201323959784
discovery of a potent benzoxaborole-based anti-pneumococcal agent targeting leucyl-trna synthetase.streptococcus pneumoniae causes bacterial pneumonia with high mortality and morbidity. the emergency of multidrug-resistant bacteria threatens the treatment of the disease. leucyl-trna synthetase (leurs) plays an essential role in cellular translation and is an attractive drug target for antimicrobial development. here we report the compound zcl039, a benzoxaborole-based derivative of an2690, as a potent anti-pneumococcal agent that inhibits s. pneumoniae leurs (spleurs) activity. we show using ...201323959225
molecular mechanisms of crispr-mediated microbial immunity.bacteriophages (phages) infect bacteria in order to replicate and burst out of the host, killing the cell, when reproduction is completed. thus, from a bacterial perspective, phages pose a persistent lethal threat to bacterial populations. not surprisingly, bacteria evolved multiple defense barriers to interfere with nearly every step of phage life cycles. phages respond to this selection pressure by counter-evolving their genomes to evade bacterial resistance. the antagonistic interaction betwe ...201323959171
molecular mechanisms of crispr-mediated microbial immunity.bacteriophages (phages) infect bacteria in order to replicate and burst out of the host, killing the cell, when reproduction is completed. thus, from a bacterial perspective, phages pose a persistent lethal threat to bacterial populations. not surprisingly, bacteria evolved multiple defense barriers to interfere with nearly every step of phage life cycles. phages respond to this selection pressure by counter-evolving their genomes to evade bacterial resistance. the antagonistic interaction betwe ...201323959171
the legionella pneumophila kai operon is implicated in stress response and confers fitness in competitive environments.legionella pneumophila uses aquatic protozoa as replication niche and protection from harsh environments. although l. pneumophila is not known to have a circadian clock, it encodes homologues of the kaibc proteins of cyanobacteria that regulate circadian gene expression. we show that l. pneumophila kaib, kaic and the downstream gene lpp1114, are transcribed as a unit under the control of the stress sigma factor rpos. kaic and kaib of l. pneumophila do not interact as evidenced by yeast and bacte ...201323957615
the legionella pneumophila kai operon is implicated in stress response and confers fitness in competitive environments.legionella pneumophila uses aquatic protozoa as replication niche and protection from harsh environments. although l. pneumophila is not known to have a circadian clock, it encodes homologues of the kaibc proteins of cyanobacteria that regulate circadian gene expression. we show that l. pneumophila kaib, kaic and the downstream gene lpp1114, are transcribed as a unit under the control of the stress sigma factor rpos. kaic and kaib of l. pneumophila do not interact as evidenced by yeast and bacte ...201323957615
one number does not fit all: mapping local variations in resolution in cryo-em reconstructions.the resolution of density maps from single particle analysis is usually measured in terms of the highest spatial frequency to which consistent information has been obtained. this calculation represents an average over the entire reconstructed volume. in practice, however, substantial local variations in resolution may occur, either from intrinsic properties of the specimen or for technical reasons such as a non-isotropic distribution of viewing orientations. to address this issue, we propose the ...201323954653
hexamers of the type ii secretion atpase gspe from vibrio cholerae with increased atpase activity.the type ii secretion system (t2ss), a multiprotein machinery spanning two membranes in gram-negative bacteria, is responsible for the secretion of folded proteins from the periplasm across the outer membrane. the critical multidomain t2ss assembly atpase gspe(epse) had not been structurally characterized as a hexamer. here, four hexamers of vibrio cholerae gspe(epse) are obtained when fused to hcp1 as an assistant hexamer, as shown with native mass spectrometry. the enzymatic activity of the gs ...201323954505
novel extracellular phb depolymerase from streptomyces ascomycinicus: phb copolymers degradation in acidic conditions.the ascomycin-producer strain streptomyces ascomycinicus has been proven to be an extracellular poly(r)-3-hydroxybutyrate (phb) degrader. the fkbu gene, encoding a phb depolymerase (phaz sa ), has been cloned in e. coli and rhodococcus sp. t104 strains for gene expression. gram-positive host rhodococcus sp. t104 was able to produce and secrete to the extracellular medium an active protein form. phaz sa was purified by two hydrophobic interaction chromatographic steps, and afterwards was biochemi ...201323951224
a comparison of structural and evolutionary attributes of escherichia coli and thermus thermophilus small ribosomal subunits: signatures of thermal adaptation.here we compare the structural and evolutionary attributes of thermus thermophilus and escherichia coli small ribosomal subunits (ssu). our results indicate that with few exceptions, thermophilic 16s ribosomal rna (16s rrna) is densely packed compared to that of mesophilic at most of the analogous spatial regions. in addition, we have located species-specific cavity clusters (ssccs) in both species. e. coli ssccs are numerous and larger compared to t. thermophilus ssccs, which again indicates de ...201323940533
nanometer scale pores similar in size to the entrance of the ribosomal exit cavity are a common feature of large rnas.the highly conserved peptidyl transferase center (ptc) of the ribosome contains an rna pore that serves as the entrance to the exit tunnel. analysis of available ribosome crystal structures has revealed the presence of multiple additional well-defined pores of comparable size in the ribosomal (rrna) rnas. these typically have dimensions of 1-2 nm, with a total area of ∼100 å(2) or more, and most are associated with one or more ribosomal proteins. the ptc example and the other rrna pores result f ...201323940386
toward repurposing ciclopirox as an antibiotic against drug-resistant acinetobacter baumannii, escherichia coli, and klebsiella pneumoniae.antibiotic-resistant infections caused by gram-negative bacteria are a major healthcare concern. repurposing drugs circumvents the time and money limitations associated with developing new antimicrobial agents needed to combat these antibiotic-resistant infections. here we identified the off-patent antifungal agent, ciclopirox, as a candidate to repurpose for antibiotic use. to test the efficacy of ciclopirox against antibiotic-resistant pathogens, we used a curated collection of acinetobacter b ...201323936064
diagnosis of barmah forest virus infection by a nested real-time sybr green rt-pcr assay.barmah forest virus (bfv) is a mosquito borne (+) ssrna alphavirus found only in australia. it causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. we developed a real-time pcr assay to detect bfv in an effort to improve diagnosis early in the course of infection. the limit of detection was 16 genome equivalents with a specificity of 100%. fifty five serum samples from bfv-infected patients were tested by the pcr. 52 of 53 antibody-positive samples were pcr negat ...201323935816
tagetitoxin inhibits transcription by stabilizing pre-translocated state of the elongation complex.transcription elongation consists of repetition of the nucleotide addition cycle: phosphodiester bond formation, translocation and binding of the next nucleotide. inhibitor of multi-subunit rna polymerase tagetitoxin (tgt) enigmatically slows down addition of nucleotides in a sequence-dependent manner, only at certain positions of the template. here, we show that tgt neither affects chemistry of rna synthesis nor induces backward translocation, nor competes with the nucleoside triphosphate (ntp) ...201323935117
an automated approach to network features of protein structure ensembles.network theory applied to protein structures provides insights into numerous problems of biological relevance. the explosion in structural data available from pdb and simulations establishes a need to introduce a standalone-efficient program that assembles network concepts/parameters under one hood in an automated manner. herein, we discuss the development/application of an exhaustive, user-friendly, standalone program package named psn-ensemble, which can handle structural ensembles generated t ...201323934896
energetic and structural details of the trigger-loop closing transition in rna polymerase ii.an evolutionarily conserved element in rna polymerase ii, the trigger loop (tl), has been suggested to play an important role in the elongation rate, fidelity of selection of the matched nucleoside triphosphate (ntp), catalysis of transcription elongation, and translocation in both eukaryotes and prokaryotes. in response to ntp binding, the tl undergoes large conformational changes to switch between distinct open and closed states to tighten the active site and avail catalysis. a computational s ...201323931324
type iv pilus proteins form an integrated structure extending from the cytoplasm to the outer membrane.the bacterial type iv pilus (t4p) is the strongest biological motor known to date as its retraction can generate forces well over 100 pn. myxococcus xanthus, a δ-proteobacterium, provides a good model for t4p investigations because its social (s) gliding motility is powered by t4p. in this study, the interactions among m. xanthus t4p proteins were investigated using genetics and the yeast two-hybrid (y2h) system. our genetic analysis suggests that there is an integrated t4p structure that crosse ...201323922942
rnase j participates in a pentatricopeptide repeat protein-mediated 5' end maturation of chloroplast mrnas.nucleus-encoded ribonucleases and rna-binding proteins influence chloroplast gene expression through their roles in rna maturation and stability. one mechanism for mrna 5' end maturation posits that sequence-specific pentatricopeptide repeat (ppr) proteins define termini by blocking the 5'→3' exonucleolytic activity of ribonuclease j (rnase j). to test this hypothesis in vivo, virus-induced gene silencing was used to reduce the expression of three ppr proteins and rnase j, both individually and ...201323921629
confounders of mutation-rate estimators: selection and phenotypic lag in thermus thermophilus.in a recent description of the rate and character of spontaneous mutation in the hyperthermophilic bacterium thermus thermophilus, the mutation rate was observed to be substantially lower than seen in several mesophiles. subsequently, a report appeared indicating that this bacterium maintains an average of about 4.5 genomes per cell. this number of genomes might result in a segregation lag for the expression of a recessive mutation and might therefore lead to an underestimate of the rate of muta ...201323916418
molecular basis of hhq biosynthesis: molecular dynamics simulations, enzyme kinetic and surface plasmon resonance studies.pqs (pseudomonasquinolone signal) and its precursor hhq are signal molecules of the p. aeruginosa quorum sensing system. they explicate their role in mammalian pathogenicity by binding to the receptor pqsr that induces virulence factor production and biofilm formation. the enzyme pqsd catalyses the biosynthesis of hhq.201323916145
pioglitazone leads to an inactivation and disassembly of complex i of the mitochondrial respiratory chain.thiazolidinediones are antidiabetic agents that increase insulin sensitivity but reduce glucose oxidation, state 3 respiration, and activity of complex i of the mitochondrial respiratory chain (mrc). the mechanisms of the latter effects are unclear. the aim of this study was to determine the mechanisms by which pioglitazone (pgz), a member of the thiazolidinedione class of antidiabetic agents, decreases the activity of the mrc. in isolated mitochondria from mouse liver, we measured the effects o ...201323915000
crystallization and preliminary x-ray analysis of an alanine dehydrogenase from bacillus megaterium wsh-002.alanine dehydrogenase (l-aladh) from bacillus megaterium wsh-002 catalyses the nad⁺-dependent interconversion of l-alanine and pyruvate. the enzyme was expressed in escherichia coli bl21 (de3) cells and purified with a his6 tag by ni²⁺-chelating affinity chromatography for x-ray crystallographic analysis. crystals were grown in a solution consisting of 0.1 m hepes ph 8.0, 12%(w/v) polyethylene glycol 8000, 8%(v/v) ethylene glycol at a concentration of 15 mg ml⁻¹ purified protein. the crystal dif ...201323908047
the putative small terminase from the thermophilic dsdna bacteriophage g20c is a nine-subunit oligomer.the assembly of double-stranded dna bacteriophages is dependent on a small terminase protein that normally plays two important roles. firstly, the small terminase protein specifically recognizes viral dna and recruits the large terminase protein, which makes the initial cut in the dsdna. secondly, once the complex of the small terminase, the large terminase and the dna has docked to the portal protein, and dna translocation into a preformed empty procapsid has begun, the small terminase modulate ...201323908032
structure of dihydrouridine synthase c (dusc) from escherichia coli.dihydrouridine (d) is one of the most widely conserved trna modifications. dihydrouridine synthase (dus) is responsible for introducing d modifications into rna by the reduction of uridine. recently, a unique substrate-recognition mechanism using a small adapter molecule has been proposed for thermus thermophilus dus (tthdusc). to acquire insight regarding its substrate-recognition mechanism, the crystal structure of dusc from escherichia coli (ecodusc) was determined at 2.1 å resolution. ecodus ...201323908023
combining crystallography and epr: crystal and solution structures of the multidomain cochaperone dnaj.hsp70 chaperones assist in a large variety of protein-folding processes in the cell. crucial for these activities is the regulation of hsp70 by hsp40 cochaperones. dnaj, the bacterial homologue of hsp40, stimulates atp hydrolysis by dnak (hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. the first structure of a complete functional dimeric dnaj was determined and the mobility of its individual domains in solution was investigated. ...201323897477
proline dehydrogenase regulates redox state and respiratory metabolism in trypanosoma cruzi.over the past three decades, l-proline has become recognized as an important metabolite for trypanosomatids. it is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. in addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. in this paper, we demonstrate that l-proline is oxid ...201323894476
proteogenomic analysis of a thermophilic bacterial consortium adapted to deconstruct switchgrass.thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. however, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. near-complet ...201323894306
crystal structure of saccharomyces cerevisiae aro8, a putative α-aminoadipate aminotransferase.α-aminoadipate aminotransferase (aaa-at) catalyzes the amination of 2-oxoadipate to α-aminoadipate in the fourth step of the α-aminoadipate pathway of lysine biosynthesis in fungi. the aromatic aminotransferase aro8 has recently been identified as an aaa-at in saccharomyces cerevisiae. this enzyme displays broad substrate selectivity, utilizing several amino acids and 2-oxo acids as substrates. here we report the 1.91å resolution crystal structure of aro8 and compare it to aaa-at lysn from therm ...201323893908
mutants of phage bil67 ruvc with enhanced holliday junction binding selectivity and resolution symmetry.viral and bacterial holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched dna substrates, while the latter exclusively targets holliday junctions. we have determined the crystal structure of a ruvc resolvase from bacteriophage bil67 to help identify features responsible for dna branch discrimination. comparisons between phage and bacterial ruvc structures revealed significant differences in the number and position of ...201323888987
genome sequence of thermus thermophilus atcc 33923, a thermostable trehalose-producing strain.thermus thermophilus atcc 33923 contains a thermostable enzyme that can efficiently catalyze the conversion of maltose into trehalose. here we report a 2.15-mb assembly of its genome sequence and other useful information, including the coding sequences (cds) responsible for biological processes such as dna replication, dna repair, and rna maturation.201323887916
the ribosome triggers the stringent response by rela via a highly distorted trna.the bacterial stringent response links nutrient starvation with the transcriptional control of genes. this process is initiated by the stringent factor rela, which senses the presence of deacylated trna in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppgpp. here we report a cryo-em study of rela bound to ribosomes bearing cognate, deacylated trna in the a-site. the data show that rela on the ribosome stabilizes an unusual distorted form of the trna, with the a ...201323877429
structural analysis of mitochondrial mutations reveals a role for bigenomic protein interactions in human disease.mitochondria are the energy producing organelles of the cell, and mutations within their genome can cause numerous and often severe human diseases. at the heart of every mitochondrion is a set of five large multi-protein machines collectively known as the mitochondrial respiratory chain (mrc). this cellular machinery is central to several processes important for maintaining homeostasis within cells, including the production of atp. the mrc is unique due to the bigenomic origin of its interacting ...201323874847
crucial optimization of translational components towards efficient incorporation of unnatural amino acids into proteins in mammalian cells.the ability to site-specifically incorporate unnatural amino acids (uaas) into proteins is a powerful tool in protein engineering. while dozens of uaas have been successfully introduced into proteins expressed by escherichia coli cells, it has been much more challenging to create trna and trna-synthetase pairs that enable uaas incorporation, for use in mammalian systems. by altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in e. coli could be use ...201323874413
genome-wide transcriptional profiling of the purple sulfur bacterium allochromatium vinosum dsm 180t during growth on different reduced sulfur compounds.the purple sulfur bacterium allochromatium vinosum dsm 180(t) is one of the best-studied sulfur-oxidizing anoxygenic phototrophic bacteria, and it has been developed into a model organism for laboratory-based studies of oxidative sulfur metabolism. here, we took advantage of the organism's high metabolic versatility and performed whole-genome transcriptional profiling to investigate the response of a. vinosum cells upon exposure to sulfide, thiosulfate, elemental sulfur, or sulfite compared to p ...201323873913
physiological role of acyl coenzyme a synthetase homologs in lipid metabolism in neurospora crassa.acyl coenzyme a (coa) synthetase (acs) enzymes catalyze the activation of free fatty acids (fas) to coa esters by a two-step thioesterification reaction. activated fas participate in a variety of anabolic and catabolic lipid metabolic pathways, including de novo complex lipid biosynthesis, fa β-oxidation, and lipid membrane remodeling. analysis of the genome sequence of the filamentous fungus neurospora crassa identified seven putative fatty acss (acs-1 through acs-7). acs-3 was found to be the ...201323873861
evidence of direct complementary interactions between messenger rnas and their cognate proteins.recently, the ability to interact with messenger rna (mrna) has been reported for a number of known rna-binding proteins, but surprisingly also for different proteins without recognizable rna binding domains including several transcription factors and metabolic enzymes. moreover, direct binding to cognate mrnas has been detected for multiple proteins, thus creating a strong impetus to search for functional significance and basic physico-chemical principles behind such interactions. here, we deri ...201323868089
the catalytic domain of topological knot trna methyltransferase (trmh) discriminates between substrate trna and nonsubstrate trna via an induced-fit process.a conserved guanosine at position 18 (g18) in the d-loop of trnas is often modified to 2'-o-methylguanosine (gm). formation of gm18 in eubacterial trna is catalyzed by trna (gm18) methyltransferase (trmh). trmh enzymes can be divided into two types based on their substrate trna specificity. type i trmh, including thermus thermophilus trmh, can modify all trna species, whereas type ii trmh, for example escherichia coli trmh, modifies only a subset of trna species. our previous crystal study showe ...201323867454
comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea.metal sulfide mineral dissolution during bioleaching and acid mine drainage (amd) formation creates an environment that is inhospitable to most life. despite dominance by a small number of bacteria, amd microbial biofilm communities contain a notable variety of coexisting and closely related euryarchaea, most of which have defied cultivation efforts. for this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring amd arch ...201323865623
energy transducing roles of antiporter-like subunits in escherichia coli ndh-1 with main focus on subunit nuon (nd2).the proton-translocating nadh-quinone oxidoreductase (complex i/ndh-1) contains a peripheral and a membrane domain. three antiporter-like subunits in the membrane domain, nuol, nuom, and nuon (nd5, nd4 and nd2, respectively), are structurally similar. we analyzed the role of nuon in escherichia coli ndh-1. the lysine residue at position 395 in nuon (nlys(395)) is conserved in nuol (llys(399)) but is replaced by glutamic acid (mglu(407)) in nuom. our mutation study on nlys(395) suggests that this ...201323864658
subunit δ is the key player for assembly of the h(+)-translocating unit of escherichia coli f(o)f1 atp synthase.the atp synthase (f(o)f1) of escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of atp. this nanomotor is composed of the rotor c10γε and the stator ab2α3β3δ. to study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled f(o)f1 complexes by aff ...201323864656
mechanism of activation of elongation factor tu by ribosome: catalytic histidine activates gtp by protonation.elongation factor tu (ef-tu) is central to prokaryotic protein synthesis as it has the role of delivering amino-acylated trnas to the ribosome. release of ef-tu, after correct binding of the ef-tu:aa-trna complex to the ribosome, is initiated by gtp hydrolysis. this reaction, whose mechanism is uncertain, is catalyzed by ef-tu, but requires activation by the ribosome. there have been a number of mechanistic proposals, including those spurred by a recent x-ray crystallographic analysis of a ribos ...201323864225
translocation and fidelity of escherichia coli rna polymerase.exonuclease (exo) iii was used as a probe of the escherichia coli rna polymerase (rnap) ternary elongation complex (tec) downstream border. in the absence of ntps, rnap appears to stall primarily in a post-translocated state and to return slowly to a pre-translocated state. exo iii mapping, therefore, appears inconsistent with an unrestrained thermal ratchet model for translocation, in which rnap freely and rapidly oscillates between pre- and post-translocated positions. the forward translocatio ...201323863783
pathway-based screening strategy for multitarget inhibitors of diverse proteins in metabolic pathways.many virtual screening methods have been developed for identifying single-target inhibitors based on the strategy of "one-disease, one-target, one-drug". the hit rates of these methods are often low because they cannot capture the features that play key roles in the biological functions of the target protein. furthermore, single-target inhibitors are often susceptible to drug resistance and are ineffective for complex diseases such as cancers. therefore, a new strategy is required for enriching ...201323861662
an innate twist between crick's wobble and watson-crick base pairs.non-watson-crick pairs like the g·u wobble are frequent in rna duplexes. their geometric dissimilarity (nonisostericity) with the watson-crick base pairs and among themselves imparts structural variations decisive for biological functions. through a novel circular representation of base pairs, a simple and general metric scheme for quantification of base-pair nonisostericity, in terms of residual twist and radial difference that can also envisage its mechanistic effect, is proposed. the scheme i ...201323861536
replacing sulfa drugs with novel dhps inhibitors.more research effort needs to be invested in antimicrobial drug development to address the increasing threat of multidrug-resistant organisms. the enzyme dhps has been a validated drug target for over 70 years as the target for the highly successful sulfa drugs. the use of sulfa drugs has been compromised by the widespread presence of resistant organisms and the adverse side effects associated with their use. despite the large amount of structural information available for dhps, few recent publi ...201323859210
structure and function of card, an essential mycobacterial transcription factor.card, an essential transcription regulator in mycobacterium tuberculosis, directly interacts with the rna polymerase (rnap). we used a combination of in vivo and in vitro approaches to establish that card is a global regulator that stimulates the formation of rnap-holoenzyme open promoter (rpo) complexes. we determined the x-ray crystal structure of thermus thermophilus card, allowing us to generate a structural model of the card/rpo complex. on the basis of our structural and functional analyse ...201323858468
characterization of the nitric oxide reductase from thermus thermophilus.nitrous oxide (n2o) is a powerful greenhouse gas implicated in climate change. the dominant source of atmospheric n2o is incomplete biological dentrification, and the enzymes responsible for the release of n2o are no reductases. it was recently reported that ambient emissions of n2o from the great boiling spring in the united states great basin are high, and attributed to incomplete denitrification by thermus thermophilus and related bacterial species [hedlund bp, et al. (2011) geobiology 9(6)47 ...201323858452
the deoxynucleotide triphosphohydrolase samhd1 is a major regulator of dna precursor pools in mammalian cells.sterile alpha motif and hd-domain containing protein 1 (samhd1) is a triphosphohydrolase converting deoxynucleoside triphosphates (dntps) to deoxynucleosides. the enzyme was recently identified as a component of the human innate immune system that restricts hiv-1 infection by removing dntps required for viral dna synthesis. samhd1 has deep evolutionary roots and is ubiquitous in human organs. here we identify a general function of samhd1 in the regulation of dntp pools in cultured human cells. t ...201323858451
bioinformatic screening of autoimmune disease genes and protein structure prediction with fams for drug discovery.autoimmune diseases are often intractable because their causes are unknown. identifying which genes contribute to these diseases may allow us to understand the pathogenesis, but it is difficult to determine which genes contribute to disease. recently, epigenetic information has been considered to activate/deactivate disease-related genes. thus, it may also be useful to study epigenetic information that differs between healthy controls and patients with autoimmune disease. among several types of ...201423855671
crystal structure of the ligand-binding form of nanornase from bacteroides fragilis, a member of the dhh/dhha1 phosphoesterase family of proteins.nanornase (nrn) specifically degrades nucleoside 3',5'-bisphosphate and the very short rna, nanorna, during the final step of mrna degradation. the crystal structure of nrn in complex with a reaction product gmp was determined. the overall structure consists of two domains that are interconnected by a flexible loop and form a cleft. two mn²⁺ ions are coordinated by conserved residues in the dhh motif of the n-terminal domain. gmp binds near the dhha1 motif region in the c-terminal domain. our st ...201323851074
sensitive and specific quantitative detection of rotavirus a by one-step real-time reverse transcription-pcr assay without antecedent double-stranded-rna denaturation.a real-time quantitative reverse transcription-pcr (qrt-pcr) assay using the recombinant thermostable thermus thermophilus (rtth) enzyme was developed to detect and quantify rotavirus a (rva). by using rtth polymerase, significant improvement was achieved over the existing real-time rt-pcr assays, which require denaturation of the rva double-stranded rna (dsrna) prior to assay setup. using a dsrna transcript for segment 7, which encodes the assay target nsp3 gene, the limit of detection for the ...201323850952
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