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the rna polymerase trigger loop functions in all three phases of the transcription cycle.the trigger loop (tl) forms a conserved element in the rna polymerase active centre that functions in the elongation phase of transcription. here, we show that the tl also functions in transcription initiation and termination. using recombinant variants of rna polymerase from pyrococcus furiosus and a reconstituted transcription system, we demonstrate that the tl is essential for initial rna synthesis until a complete dna-rna hybrid is formed. the archaeal tl is further important for transcripti ...201323737452
thermal and spectroscopic characterization of a proton pumping rhodopsin from an extreme thermophile.so far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. in this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium thermus thermophilus, which lives in a hot spring at around 75 °c. the gene for the retinylidene protein, named thermophilic rhodopsin (tr), was chemically synthesized with codon optimization. the codon optimized tr protein was functionally expressed in the cell membranes of escherichia ...201323740255
structural and functional insight into the universal stress protein family.we present the crystal structures of two universal stress proteins (usp) from archaeoglobus fulgidus and nitrosomonas europaea in both apo- and ligand-bound forms. this work is the first complete synthesis of the structural properties of 26 usp available in the protein data bank, over 75% of which were determined by structure genomics centers with no additional information provided. the results of bioinformatic analyses of all available usp structures and their sequence homologs revealed that th ...201323745136
methylerythritol phosphate pathway of isoprenoid biosynthesis.isoprenoids are a class of natural products with more than 55,000 members. all isoprenoids are constructed from two precursors, isopentenyl diphosphate and its isomer dimethylallyl diphosphate. two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway [the methylerythritol phosphate (mep) pathway] and a modified mevalonic acid (mva) pathway. in this review, we summarize mechanistic insights on the mep ...201323746261
the making of a slicer: activation of human argonaute-1.argonautes are the central protein component in small rna silencing pathways. of the four human argonautes (hago1-hago4) only hago2 is an active slicer. we determined the structure of hago1 bound to endogenous copurified rnas to 1.75 å resolution and hago1 loaded with let-7 microrna to 2.1 å. both structures are strikingly similar to the structures of hago2. a conserved catalytic tetrad within the piwi domain of hago2 is required for its slicing activity. completion of the tetrad, combined with ...201323746446
structural determinants of oligomerization of δ(1)-pyrroline-5-carboxylate dehydrogenase: identification of a hexamerization hot spot.the aldehyde dehydrogenase (aldh) superfamily member δ(1)-pyrroline-5-carboxylate dehydrogenase (p5cdh) catalyzes the nad(+)-dependent oxidation of glutamate semialdehyde to glutamate, which is the final step of proline catabolism. defects in p5cdh activity lead to the metabolic disorder type ii hyperprolinemia, p5cdh is essential for virulence of the fungal pathogen cryptococcus neoformans, and bacterial p5cdhs have been targeted for vaccine development. although the enzyme oligomeric state is ...201323747974
lack of discrimination against non-proteinogenic amino acid norvaline by elongation factor tu from escherichia coli.the gtp-bound form of elongation factor tu (ef-tu) brings aminoacylated trnas (aa-trna) to the a-site of the ribosome. ef-tu binds all cognate elongator aa-trnas with highly similar affinities, and its weaker or tighter binding of misacylated trnas may discourage their participation in translation. norvaline (nva) is a non-proteinogenic amino acid that is activated and transferred to trna(leu) by leucyl-trna synthetase (leurs). no notable accumulation of nva-trna(leu) has been observed in vitro, ...201323750044
correlation spectroscopy and molecular dynamics simulations to study the structural features of proteins.in this work, we used a combination of fluorescence correlation spectroscopy (fcs) and molecular dynamics (md) simulation methodologies to acquire structural information on ph-induced unfolding of the maltotriose-binding protein from thermus thermophilus (male2). fcs has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. our results showed that keeping temperature constant, the ...201323750215
distinction between the cfr methyltransferase conferring antibiotic resistance and the housekeeping rlmn methyltransferase.the cfr gene encodes the cfr methyltransferase that primarily methylates c-8 in a2503 of 23s rrna in the peptidyl transferase region of bacterial ribosomes. the methylation provides resistance to six classes of antibiotics of clinical and veterinary importance. the rlmn gene encodes the rlmn methyltransferase that methylates c-2 in a2503 in 23s rrna and a37 in trna, but rlmn does not significantly influence antibiotic resistance. the enzymes are homologous and use the same mechanism involving ra ...201323752511
structure and function of allophanate hydrolase.allophanate hydrolase converts allophanate to ammonium and carbon dioxide. it is conserved in many organisms and is essential for their utilization of urea as a nitrogen source. it also has important functions in a newly discovered eukaryotic pyrimidine nucleic acid precursor degradation pathway, the yeast-hypha transition that several pathogens utilize to escape the host defense, and an s-triazine herbicide degradation pathway recently emerged in many soil bacteria. we have determined the cryst ...201323754281
structure and function of the t-loop structural motif in noncoding rnas.the t-loop is a frequently occurring five-nucleotide motif found in the structure of noncoding rnas where it is commonly assumed to play an important role in stabilizing the tertiary rna structure by facilitating long-range interactions between different regions of the molecule. t-loops were first identified in trna(phe) and a formal consensus sequence for this motif was formulated and later revised based on analyses of the crystal structures of prokaryotic ribosomal rnas and rnase p and the cor ...201323754657
energy, genes and evolution: introduction to an evolutionary synthesis.life is the harnessing of chemical energy in such a way that the energy-harnessing device makes a copy of itself. no energy, no evolution. the 'modern synthesis' of the past century explained evolution in terms of genes, but this is only part of the story. while the mechanisms of natural selection are correct, and increasingly well understood, they do little to explain the actual trajectories taken by life on earth. from a cosmic perspective-what is the probability of life elsewhere in the unive ...201323754807
in vitro reconstitution of an escherichia coli rna-guided immune system reveals unidirectional, atp-dependent degradation of dna target.many prokaryotes utilize small rna transcribed from clustered, regularly interspaced, short palindromic repeats (crisprs) to protect themselves from foreign genetic elements, such as phage and plasmids. in escherichia coli, this small rna is packaged into a surveillance complex (cascade) that uses the rna sequence to direct binding to invasive dna. once bound, cascade recruits the cas3 nuclease-helicase, which then proceeds to progressively degrade the invading dna. here, using individually puri ...201323760266
molecular crowding enhanced atpase activity of the rna helicase eif4a correlates with compaction of its quaternary structure and association with eif4g.enzymatic reactions occur in a crowded and confined environment in vivo, containing proteins, rna and dna. previous reports have shown that interactions between macromolecules, and reactions rates differ significantly between crowded environments and dilute buffers. however, the direct effect of crowding on the level of high-resolution structures of macromolecules has not been extensively analyzed and is not well understood. here we analyze the effect of macromolecular crowding on structure and ...201323767688
crystal structure of the 70s ribosome bound with the q253p mutant form of release factor rf2.bacterial translation termination is mediated by release factors rf1 and rf2, which recognize stop codons and catalyze hydrolysis of the peptidyl-trna ester bond. the catalytic mechanism has been debated. we proposed that the backbone amide nh group, rather than the side chain, of the glutamine of the universally conserved ggq motif participates in catalysis by h-bonding to the tetrahedral transition-state intermediate and by product stabilization. this was supported by complete loss of rf1 cata ...201323769667
antituberculosis thiophenes define a requirement for pks13 in mycolic acid biosynthesis.we report a new class of thiophene (tp) compounds that kill mycobacterium tuberculosis by the previously uncharacterized mechanism of pks13 inhibition. an f79s mutation near the catalytic ser55 site in pks13 conferred tp resistance in m. tuberculosis. overexpression of wild-type pks13 resulted in tp resistance, and overexpression of the pks13(f79s) mutant conferred high resistance. in vitro, tp inhibited fatty acyl-amp loading onto pks13. tp inhibited mycolic acid biosynthesis in wild-type m. tu ...201323770708
secondary structure and domain architecture of the 23s and 5s rrnas.we present a de novo re-determination of the secondary (2°) structure and domain architecture of the 23s and 5s rrnas, using 3d structures, determined by x-ray diffraction, as input. in the traditional 2° structure, the center of the 23s rrna is an extended single strand, which in 3d is seen to be compact and double helical. accurately assigning nucleotides to helices compels a revision of the 23s rrna 2° structure. unlike the traditional 2° structure, the revised 2° structure of the 23s rrna sh ...201323771137
zinc-binding and structural properties of the histidine-rich loop of arabidopsis thaliana vacuolar membrane zinc transporter mtp1.the vacuolar zn(2+)/h(+) antiporter of arabidopsis thaliana, atmtp1, has a cytosolic histidine-rich loop (his-loop). we characterized the structures and zn(2+)-binding properties of the his-loop and other domains. circular dichroism analyses revealed that the his-loop partly consists of a polyproline type ii structure and that its conformational change is induced by zn(2+) as well as the c-terminal domain. isothermal titration calorimetry of the his-loop revealed a binding number of four zn(2+) ...201323772397
dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates.the efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. we used dna-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both zymomonas mobilis (44 genes) and saccharomyces cerevisiae (99 genes). overexpression of a z. mobilis tolerance gene of unknown functi ...201323774757
coordination between apoplastic and symplastic detoxification confers plant aluminum resistance.whether aluminum toxicity is an apoplastic or symplastic phenomenon is still a matter of debate. here, we found that three auxin overproducing mutants, yucca, the recessive mutant superroot2, and superroot1 had increased aluminum sensitivity, while a transfer dna insertion mutant, xyloglucan endotransglucosylase/hydrolases15 (xth15), showed enhanced aluminum resistance, accompanied by low endogenous indole-3-acetic acid levels, implying that auxin may be involved in plant responses to aluminum s ...201323776189
experimental evidence for the thermophilicity of ancestral life.theoretical studies have focused on the environmental temperature of the universal common ancestor of life with conflicting conclusions. here we provide experimental support for the existence of a thermophilic universal common ancestor. we present the thermal stabilities and catalytic efficiencies of nucleoside diphosphate kinases (ndk), designed using the information contained in predictive phylogenetic trees, that seem to represent the last common ancestors of archaea and of bacteria. these en ...201323776221
expanded use of sense codons is regulated by modified cytidines in trna.codon use among the three domains of life is not confined to the universal genetic code. with only 22 trna genes in mammalian mitochondria, exceptions from the universal code are necessary for proper translation. a particularly interesting deviation is the decoding of the isoleucine aua codon as methionine by the one mitochondrial-encoded trna(met). this trna decodes aua and aug in both the a- and p-sites of the metazoan mitochondrial ribosome. enrichment of posttranscriptional modifications is ...201323781103
the role of mitochondrial function and cellular bioenergetics in ageing and disease.mitochondria constitute an important topic of biomedical enquiry (one paper in every 154 indexed in pubmed since 1998 is retrieved by the keyword 'mitochondria') because of widespread recognition of their importance in cell physiology and pathology. mitochondrial dysfunction is widely implicated in ageing and in the diseases of ageing, through dysfunction in adenosine triphosphate (atp) synthesis, ca(2+) homeostasis, central metabolic pathways or radical production. nonetheless, the mechanisms a ...201323786614
polysaccharide-degrading thermophiles generated by heterologous gene expression in geobacillus kaustophilus hta426.thermophiles have important advantages over mesophiles as host organisms for high-temperature bioprocesses, functional production of thermostable enzymes, and efficient expression of enzymatic activities in vivo. to capitalize on these advantages of thermophiles, we describe here a new inducible gene expression system in the thermophile geobacillus kaustophilus hta426. six promoter regions in the hta426 genome were identified and analyzed for expression profiles using β-galactosidase reporter as ...201323793634
two atp-binding cassette transporters involved in (s)-2-aminoethyl-cysteine uptake in thermus thermophilus.thermus thermophilus exhibits hypersensitivity to a lysine analog, (s)-2-aminoethyl-cysteine (aec). cosmid libraries were constructed using genomes from two aec-resistant mutants, at10 and at14, and the cosmids that conferred aec resistance on the wild-type strain were isolated. when the cosmid library for mutant at14 was screened, two independent cosmids, conferring partial aec resistance to the wild type, were obtained. two cosmids carried a common genomic region from ttc0795 to ttc0810. this ...201323794618
development of a d-xylose fermenting and inhibitor tolerant industrial saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering.the production of bioethanol from lignocellulose hydrolysates requires a robust, d-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. the purpose of the present work was to develop such a strain from a prime industrial yeast strain, ethanol red, used for bioethanol production.201323800147
the trna recognition mechanism of the minimalist spout methyltransferase, trml.unlike other transfer rnas (trna)-modifying enzymes from the spout methyltransferase superfamily, the trna (um34/cm34) methyltransferase trml lacks the usual extension domain for trna binding and consists only of a spout domain. both the catalytic and trna recognition mechanisms of this enzyme remain elusive. by using trnas purified from an escherichia coli strain with the trml gene deleted, we found that trml can independently catalyze the methyl transfer from s-adenosyl-l-methionine to and iso ...201323804755
structural insight into negative dna supercoiling by dna gyrase, a bacterial type 2a dna topoisomerase.type 2a dna topoisomerases (topo2a) remodel dna topology during replication, transcription and chromosome segregation. these multisubunit enzymes catalyze the transport of a double-stranded dna through a transient break formed in another duplex. the bacterial dna gyrase, a target for broad-spectrum antibiotics, is the sole topo2a enzyme able to introduce negative supercoils. we reveal here for the first time the architecture of the full-length thermus thermophilus dna gyrase alone and in a cleav ...201323804759
structural basis for s-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor b1.eukaryotic transcription factor b (tfb) proteins are homologous to ksga/dim1 ribosomal rna (rrna) methyltransferases. the mammalian tfb1, mitochondrial (tfb1m) factor is an essential protein necessary for mitochondrial gene expression. tfb1m mediates an rrna modification in the small ribosomal subunit and thus plays a role analogous to ksga/dim1 proteins. this modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and diabe ...201323804760
co-expression of rna-protein complexes in escherichia coli and applications to rna biology.rna has emerged as a major player in many cellular processes. understanding these processes at the molecular level requires homogeneous rna samples for structural, biochemical and pharmacological studies. we previously devised a generic approach that allows efficient in vivo expression of recombinant rna in escherichia coli. in this work, we have extended this method to rna/protein co-expression. we have engineered several plasmids that allow overexpression of rna-protein complexes in e. coli. w ...201323804766
crispr interference: a structural perspective.crispr (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. genomically encoded crrna (crispr rna) is used by cas (crispr-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. the process is known as 'interference'. in the present review we cover recent progress on the structural biology of the crispr/cas system, focu ...201323805973
eukaryote-specific insertion elements control human argonaute slicer activity.we have solved the crystal structure of human argonaute1 (hago1) bound to endogenous 5'-phosphorylated guide rnas. to identify changes that evolutionarily rendered hago1 inactive, we compared our structure with guide-rna-containing and cleavage-active hago2. aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hago1 introduce a kink in the cs7 loop, forming a convex surface within the hago1 nucleic-acid-binding channel near the inactive catalytic site. ...201323809764
the role of aromatic-aromatic interactions in strand-strand stabilization of β-sheets.aromatic-aromatic interactions have long been believed to play key roles in protein structure, folding, and binding functions. however, we still lack full understanding of the contributions of aromatic-aromatic interactions to protein stability and the timing of their formation during folding. here, using an aromatic ladder in the β-barrel protein, cellular retinoic acid-binding protein 1 (crabp1), as a case study, we find that aromatic π stacking plays a greater role in the phe65-phe71 cross-st ...201323810905
unusual base pairing during the decoding of a stop codon by the ribosome.during normal translation, the binding of a release factor to one of the three stop codons (uga, uaa or uag) results in the termination of protein synthesis. however, modification of the initial uridine to a pseudouridine (ψ) allows efficient recognition and read-through of these stop codons by a transfer rna (trna), although it requires the formation of two normally forbidden purine-purine base pairs. here we determined the crystal structure at 3.1 å resolution of the 30s ribosomal subunit in c ...201323812587
elongation factor g bound to the ribosome in an intermediate state of translocation.a key step of translation by the ribosome is translocation, which involves the movement of messenger rna (mrna) and transfer rna (trna) with respect to the ribosome. this allows a new round of protein chain elongation by placing the next mrna codon in the a site of the 30s subunit. translocation proceeds through an intermediate state in which the acceptor ends of the trnas have moved with respect to the 50s subunit but not the 30s subunit, to form hybrid states. the guanosine triphosphatase (gtp ...201323812720
crystal structures of ef-g-ribosome complexes trapped in intermediate states of translocation.translocation of messenger and transfer rna (mrna and trna) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. the crystal structures of three thermus ribosome-trna-mrna-ef-g complexes trapped with β,γ-imidoguanosine 5'-triphosphate (gdpnp) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30s subunit head and body. in all complexes, the trna acceptor ends occ ...201323812722
by ribosome possessed. 201323814064
an 'open' structure of the recor complex supports ssdna binding within the core of the complex.efficient dna repair is critical for cell survival and the maintenance of genome integrity. the homologous recombination pathway is responsible for the repair of dna double-strand breaks within cells. initiation of this pathway in bacteria can be carried out by either the recbcd or the recfor proteins. an important regulatory player within the recfor pathway is the recor complex that facilitates reca loading onto dna. here we report new data regarding the assembly of deinococcus radiodurans reco ...201323814185
an insertion peptide in yeast glycyl-trna synthetase facilitates both productive docking and catalysis of cognate trnas.the yeast saccharomyces cerevisiae possesses two distinct glycyl-trna synthetase (glyrs) genes: grs1 and grs2. grs1 is dually functional, encoding both cytoplasmic and mitochondrial activities, while grs2 is dysfunctional and not required for growth. the protein products of these two genes, glyrs1 and glyrs2, are much alike but are distinguished by an insertion peptide of glyrs1, which is absent from glyrs2 and other eukaryotic homologues. we show that deletion or mutation of the insertion pepti ...201323816885
biology of extreme radiation resistance: the way of deinococcus radiodurans.the bacterium deinococcus radiodurans is a champion of extreme radiation resistance that is accounted for by a highly efficient protection against proteome, but not genome, damage. a well-protected functional proteome ensures cell recovery from extensive radiation damage to other cellular constituents by molecular repair and turnover processes, including an efficient repair of disintegrated dna. therefore, cell death correlates with radiation-induced protein damage, rather than dna damage, in bo ...201323818498
crystal structure of the ubiquitin-like small archaeal modifier protein 2 from haloferax volcanii.the discovery of ubiquitin-like small archaeal modifier protein 2 (samp2) that forms covalent polymeric chains in haloferax volcanii has generated tremendous interest in the function and regulation of this protein. at present, it remains unclear whether the hfx. volcanii modifier protein samp1 has such polyubiquitinating-like activity. although samp1 and samp2 use the same conjugation machinery to modify their target proteins, each can impart distinct functional consequences. to better understan ...201323821306
blasticidin s inhibits translation by trapping deformed trna on the ribosome.the antibiotic blasticidin s (blas) is a potent inhibitor of protein synthesis in bacteria and eukaryotes. we have determined a 3.4-å crystal structure of blas bound to a 70s⋅trna ribosome complex and performed biochemical and single-molecule fret experiments to determine the mechanism of action of the antibiotic. we find that blas enhances trna binding to the p site of the large ribosomal subunit and slows down spontaneous intersubunit rotation in pretranslocation ribosomes. however, the antibi ...201323824292
translation of cga codon repeats in yeast involves quality control components and ribosomal protein l1.translation of cga codon repeats in the yeast saccharomyces cerevisiae is inefficient, resulting in dose-dependent reduction in expression and in production of an mrna cleavage product, indicative of a stalled ribosome. here, we use genetics and translation inhibitors to understand how ribosomes respond to cga repeats. we find that cga codon repeats result in a truncated polypeptide that is targeted for degradation by ltn1, an e3 ubiquitin ligase involved in nonstop decay, although deletion of l ...201323825054
heritable change caused by transient transcription errors.transmission of cellular identity relies on the faithful transfer of information from the mother to the daughter cell. this process includes accurate replication of the dna, but also the correct propagation of regulatory programs responsible for cellular identity. errors in dna replication (mutations) and protein conformation (prions) can trigger stable phenotypic changes and cause human disease, yet the ability of transient transcriptional errors to produce heritable phenotypic change ('epimuta ...201323825966
phosphoproteomic analysis reveals the effects of pilf phosphorylation on type iv pilus and biofilm formation in thermus thermophilus hb27.thermus thermophilus hb27 is an extremely thermophilic eubacteria with a high frequency of natural competence. this organism is therefore often used as a thermophilic model to investigate the molecular basis of type iv pili-mediated functions, such as the uptake of free dna, adhesion, twitching motility, and biofilm formation, in hot environments. in this study, the phosphoproteome of t. thermophilus hb27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. ninety-three uniqu ...201323828892
kinetic and isotopic characterization of l-proline dehydrogenase from mycobacterium tuberculosis.the monofunctional proline dehydrogenase (prodh) from mycobacterium tuberculosis performs the flavin-dependent oxidation of l-proline to δ(1)-pyrroline-5-carboxylate in the proline catabolic pathway. the prodh gene, prub, was cloned into the pyub1062 vector, and the c-terminal his-tagged 37 kda protein was expressed and purified by nickel affinity chromatography. a steady-state kinetic analysis revealed a ping-pong mechanism with an overall kcat of 33 ± 2 s(-1) and km values of 5.7 ± 0.8 mm and ...201323834473
post-translational modifications near the quinone binding site of mammalian complex i.complex i (nadh:ubiquinone oxidoreductase) in mammalian mitochondria is an l-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 å into the matrix. the protruding arm contains the binding sites for nadh, the primary acceptor of electrons flavin mononucleotide (fmn), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from fmn to a coenzyme q molecule bound in the vicinity ...201323836892
kinetic proofreading at single molecular level: aminoacylation of trna(ile) and the role of water as an editor.proofreading/editing in protein synthesis is essential for accurate translation of information from the genetic code. in this article we present a theoretical investigation of efficiency of a kinetic proofreading mechanism that employs hydrolysis of the wrong substrate as the discriminatory step in enzyme catalytic reactions. we consider aminoacylation of trna(ile) which is a crucial step in protein synthesis and for which experimental results are now available. we present an augmented kinetic s ...201323840412
extremophilic shmts: from structure to biotechnology.recent advances in molecular and structural biology have improved the availability of virtually any biocatalyst in large quantity and have also provided an insight into the detailed structure-function relationships of many of them. these results allowed the rational exploitation of biocatalysts for use in organic synthesis. in this context, extremophilic enzymes are extensively studied for their potential interest for many biotechnological and industrial applications, as they offer increased rat ...201323841096
structure of the archaeal cascade subunit csa5: relating the small subunits of crispr effector complexes.the cascade complex for crispr-mediated antiviral immunity uses crispr rna (crrna) to target invading dna species from mobile elements such as viruses, leading to their destruction. the core of the cascade effector complex consists of the cas5 and cas7 subunits, which are widely conserved in prokaryotes. cas7 binds crrna and forms the helical backbone of cascade. many archaea encode a version of the cascade complex (denoted type i-a) that includes a csa5 (or small) subunit, which interacts weakl ...201323846216
individual interactions of the b subunits within the stator of the escherichia coli atp synthase.fof1 atp synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of atp. during catalysis, the peripheral stalk, composed of two b subunits and subunit δ in escherichia coli, counteracts the torque generated by the rotation of the central stalk. here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. ant ...201323846684
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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