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recognition and cleavage of a nonstructured crispr rna by its processing endoribonuclease cas6.clustered regularly interspaced short palindromic repeats (crisprs) confer adaptive immunity to prokaryotes through a small rna-mediated mechanism. specific endoribonucleases are required by all crispr-bearing organisms to process crispr rnas into small rna that serve as guides for defensive effector complexes. the molecular mechanism of how the endoribonucleases process the class of crispr rna containing no predicted secondary structural features remains largely elusive. here, we report cocryst ...201323454186
pilmnopq from the pseudomonas aeruginosa type iv pilus system form a transenvelope protein interaction network that interacts with pila.pseudomonas aeruginosa type iv pili (t4p) are virulence factors that promote infection of cystic fibrosis and immunosuppressed patients. as the absence of t4p impairs colonization, they are attractive targets for the development of novel therapeutics. genes in the pilmnopq operon are important for both t4p assembly and a form of bacterial movement, called twitching motility, that is required for pathogenicity. the type ii membrane proteins, piln and pilo, dimerize via their periplasmic domains a ...201323457250
an engineered heme-copper center in myoglobin: co migration and binding.we have investigated co migration and binding in cubmb, a copper-binding myoglobin double mutant (l29h-f43h), by using fourier transform infrared spectroscopy and flash photolysis over a wide temperature range. this mutant was originally engineered with the aim to mimic the catalytic site of heme-copper oxidases. comparison of the wild-type protein mb and cubmb shows that the copper ion in the distal pocket gives rise to significant effects on ligand binding to the heme iron. in mb and copper-fr ...201323459127
the variability of the 16s rrna gene in bacterial genomes and its consequences for bacterial community analyses.16s ribosomal rna currently represents the most important target of study in bacterial ecology. its use for the description of bacterial diversity is, however, limited by the presence of variable copy numbers in bacterial genomes and sequence variation within closely related taxa or within a genome. here we use the information from sequenced bacterial genomes to explore the variability of 16s rrna sequences and copy numbers at various taxonomic levels and apply it to estimate bacterial genome an ...201323460914
diversity in the origins of proteostasis networks--a driver for protein function in evolution.although the sequence of a protein largely determines its function, proteins can adopt different folding states in response to changes in the environment, some of which may be deleterious to the organism. all organisms--bacteria, archaea and eukarya--have evolved a protein homeostasis, or proteostasis, network comprising chaperones and folding factors, degradation components, signalling pathways and specialized compartmentalized modules that manage protein folding in response to environmental st ...201323463216
erythroid heme biosynthesis and its disorders.heme, which is composed of iron and the small organic molecule protoporphyrin, is an essential component of hemoglobin as well as a variety of physiologically important hemoproteins. during erythropoiesis, heme synthesis is induced before, and is essential for, globin synthesis. although all cells possess the ability to synthesize heme, there are distinct differences between regulation of the pathway in developing erythroid cells and all other types of cells. disorders that compromise the abilit ...201323471474
crystal and nmr structures give insights into the role and dynamics of subunit f of the eukaryotic v-atpase from saccharomyces cerevisiae.subunit f of v-atpases is proposed to undergo structural alterations during catalysis and reversible dissociation from the v1vo complex. recently, we determined the low resolution structure of f from saccharomyces cerevisiae v-atpase, showing an n-terminal egg shape, connected to a c-terminal hook-like segment via a linker region. to understand the mechanistic role of subunit f of s. cerevisiae v-atpase, composed of 118 amino acids, the crystal structure of the major part of f, f(1-94), was solv ...201323476018
structure of the poliiiα-τc-dna complex suggests an atomic model of the replisome.the c-terminal domain (ctd) of the τ subunit of the clamp loader (τc) binds to both the dnab helicase and the dna polymerase iii α subunit (poliiiα), and determines their relative positions and orientations on the leading and lagging strands. here, we present a 3.2 å resolution structure of thermus aquaticus poliiiα in complex with τc and a dna substrate. the structure reveals that the ctd of τc interacts with the ctd of poliiiα through its c-terminal helix and the adjacent loop. additionally, i ...201323478062
structural units important for activity of a novel-type phosphoserine phosphatase from hydrogenobacter thermophilus tk-6 revealed by crystal structure analysis.novel-type serine-synthesizing enzymes, termed metal-independent phosphoserine phosphatases (ipsps), were recently identified and characterized from hydrogenobacter thermophilus, a chemolithoautotrophic bacterium belonging to the order aquificales. ipsps are cofactor-dependent phosphoglycerate mutase (dpgm)-like phosphatases that have significant amino acid sequence similarity to dpgms but lack phosphoglycerate mutase activity. genes coding dpgm-like phosphatases have been identified in a broad ...201323479726
electron transfer mechanism of the rieske protein from thermus thermophilus from solution nuclear magnetic resonance investigations.we report nuclear magnetic resonance (nmr) data indicating that the rieske protein from the cytochrome bc complex of thermus thermophilus (ttrp) undergoes modest redox-state-dependent and ligand-dependent conformational changes. to test models concerning the mechanism by which ttrp transfers between different sites on the complex, we monitored (1)h, (15)n, and (13)c nmr signals as a function of the redox state and molar ratio of added ligand. our studies of full-length ttrp were conducted in the ...201323480240
the crispr-associated gene cas2 of legionella pneumophila is required for intracellular infection of amoebae.recent studies have shown that the clustered regularly interspaced palindromic repeats (crispr) array and its associated (cas) genes can play a key role in bacterial immunity against phage and plasmids. upon analysis of the legionella pneumophila strain 130b chromosome, we detected a subtype ii-b crispr-cas locus that contains cas9, cas1, cas2, cas4, and an array with 60 repeats and 58 unique spacers. reverse transcription (rt)-pcr analysis demonstrated that the entire crispr-cas locus is expres ...201323481601
determination of the structure of the catabolic n-succinylornithine transaminase (astc) from escherichia coli.escherichia coli possesses two acyl ornithine aminotransferases, one catabolic (astc) and the other anabolic (argd), that participate in l-arginine metabolism. although only 58% identical, the enzymes have been shown to be functionally interchangeable. here we have purified astc and have obtained x-ray crystal structures of apo and holo-astc and of the enzyme complexed with its physiological substrate, succinylornithine. we compare the structures obtained in this study with those of argd from sa ...201323484010
simultaneous quantification of mitochondrial dna damage and copy number in circulating blood: a sensitive approach to systemic oxidative stress.systemic oxidative stress is associated with a wide range of pathological conditions. oxidative dna damage is frequently measured in circulating lymphocytes. mitochondrial dna (mtdna) is known to be more sensitive to oxidative damage than nuclear dna but is rarely used for direct measurement of dna damage in clinical studies. based on the supercoiling-sensitive real-time pcr method, we propose a new approach for the noninvasive monitoring of systemic oxidative stress by quantifying the mtdna str ...201223484085
simultaneous quantification of mitochondrial dna damage and copy number in circulating blood: a sensitive approach to systemic oxidative stress.systemic oxidative stress is associated with a wide range of pathological conditions. oxidative dna damage is frequently measured in circulating lymphocytes. mitochondrial dna (mtdna) is known to be more sensitive to oxidative damage than nuclear dna but is rarely used for direct measurement of dna damage in clinical studies. based on the supercoiling-sensitive real-time pcr method, we propose a new approach for the noninvasive monitoring of systemic oxidative stress by quantifying the mtdna str ...201223484085
characterization of halomonas sp. zm3 isolated from the zelazny most post-flotation waste reservoir, with a special focus on its mobile dna.halomonas sp. zm3 was isolated from zelazny most post-flotation mineral waste repository (poland), which is highly contaminated with heavy metals and various organic compounds. mobile dna of the strain (i.e. plasmids and transposons) were analyzed in order to identify genetic information enabling adaptation of the bacterium to the harsh environmental conditions.201323497212
the role of mirnas in regulating gene expression networks.micrornas (mirnas) are key regulators of gene expression. they are conserved across species, expressed across cell types, and active against a large proportion of the transcriptome. the sequence-complementary mechanism of mirna activity exploits combinatorial diversity, a property conducive to network-wide regulation of gene expression, and functional evidence supporting this hypothesized systems-level role has steadily begun to accumulate. the emerging models are exciting and will yield deep in ...201323500488
the human flavoproteome.vitamin b2 (riboflavin) is an essential dietary compound used for the enzymatic biosynthesis of fmn and fad. the human genome contains 90 genes encoding for flavin-dependent proteins, six for riboflavin uptake and transformation into the active coenzymes fmn and fad as well as two for the reduction to the dihydroflavin form. flavoproteins utilize either fmn (16%) or fad (84%) while five human flavoenzymes have a requirement for both fmn and fad. the majority of flavin-dependent enzymes catalyze ...201323500531
uga is an additional glycine codon in uncultured sr1 bacteria from the human microbiota.the composition of the human microbiota is recognized as an important factor in human health and disease. many of our cohabitating microbes belong to phylum-level divisions for which there are no cultivated representatives and are only represented by small subunit rrna sequences. for one such taxon (sr1), which includes bacteria with elevated abundance in periodontitis, we provide a single-cell genome sequence from a healthy oral sample. sr1 bacteria use a unique genetic code. in-frame tga (opal ...201323509275
thermostable mismatch-recognizing protein muts suppresses nonspecific amplification during polymerase chain reaction (pcr).polymerase chain reaction (pcr)-related technologies are hampered mainly by two types of error: nonspecific amplification and dna polymerase-generated mutations. here, we report that both errors can be suppressed by the addition of a dna mismatch-recognizing protein, muts, from a thermophilic bacterium. although it had been expected that muts has a potential to suppress polymerase-generated mutations, we unexpectedly found that it also reduced nonspecific amplification. on the basis of this find ...201323519109
structure of vibrio cholerae ribosome hibernation promoting factor.the x-ray crystal structure of ribosome hibernation promoting factor (hpf) from vibrio cholerae is presented at 2.0 å resolution. the crystal was phased by two-wavelength mad using cocrystallized cobalt. the asymmetric unit contained two molecules of hpf linked by four co atoms. the metal-binding sites observed in the crystal are probably not related to biological function. the structure of hpf has a typical β-α-β-β-β-α fold consistent with previous structures of yfia and hpf from escherichia co ...201323519794
crystallization and preliminary x-ray analysis of peptidyl-trna hydrolase from thermus thermophilus hb8.peptidyl-trna is produced from the ribosome as a result of aborted translation. peptidyl-trna hydrolase cleaves the ester bond between the peptide and the trna of peptidyl-trna molecules, to recycle trna for further rounds of protein synthesis. in this study, peptidyl-trna hydrolase from thermus thermophilus hb8 (tthpth) was crystallized using 2-methyl-2,4-pentanediol as a precipitant. the crystals belonged to the orthorhombic space group p2₁2₁2₁, with unit-cell parameters a=47.45, b=53.92, c=58 ...201323519816
comparative proteomics identifies the cell-associated lethality of m. tuberculosis relbe-like toxin-antitoxin complexes.the mycobacterium tuberculosis (mtb) genome encodes approximately 90 toxin-antitoxin protein complexes, including three relbe family members, which are believed to play a major role in bacterial fitness and pathogenicity. we have determined the crystal structures of mtb relbe-2 and relbe-3, and the structures reveal homologous heterotetramers. our structures suggest rele-2, and by extension the closely related rele-1, use a different catalytic mechanism than rele-3, because our analysis of the r ...201323523424
camphor pathway redux: functional recombinant expression of 2,5- and 3,6-diketocamphane monooxygenases of pseudomonas putida atcc 17453 with their cognate flavin reductase catalyzing baeyer-villiger reactions.whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-dkcmo and 3,6-dkcmo, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (cam) metabolism in pseudomonas putida atcc 17453 are relatively well characterized, the actual identity of the flavin reductase (fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. in this study, a 37- ...201323524667
improved transferase/hydrolase ratio through rational design of a family 1 β-glucosidase from thermotoga neapolitana.alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very difficult to do using conventional chemistry. however, there is a need for better enzymes to get a commercially feasible process. a thermostable β-glucosidase from the well-studied glycoside hydrolase family 1 from thermotoga neapolitana, tnbgl1a, ...201323524680
leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-trna synthetase.the leucine-specific domain (lsd) is a compact well-ordered module that participates in positioning of the conserved kmsks catalytic loop in most leucyl-trna synthetases (leurss). however, the leurs from mycoplasma mobile (mmleurs) has a tetrapeptide gkdg instead of the lsd. here, we show that the tetrapeptide gkdg can confer trna charging and post-transfer editing activity when transplanted into an inactive escherichia coli leurs (ecleurs) that has had its lsd deleted. reciprocally, the lsd, to ...201323525458
structural insights into putative molybdenum cofactor biosynthesis protein c (moac2) from mycobacterium tuberculosis h37rv.the molybdenum cofactor (moco) biosynthesis pathway is an evolutionary conserved pathway seen in almost all eukaryotes including the pathogenic species mycobacterium tuberculosis. this pathway comprises of several novel reactions which include the initial formation of precursor z from guanosine triphosphate (gtp), catalysed by two enzymes moaa and moac. although moco biosynthesis is well understood, the first step is still not clear. in m. tuberculosis h37rv, three orthologous genes of moac have ...201323526978
crystal structure of 70s ribosome with both cognate trnas in the e and p sites representing an authentic elongation complex.during the translation cycle, a cognate deacylated trna can only move together with the codon into the e site. we here present the first structure of a cognate trna bound to the ribosomal e site resulting from translocation by ef-g, in which an entire l1 stalk (l1 protein and l1 rrna) interacts with e-site trna (e-trna), representing an authentic ribosome elongation complex. our results revealed that the watson-crick base pairing is formed at the first and second codon-anticodon positions in the ...201323527033
structure of a dimeric crenarchaeal cas6 enzyme with an atypical active site for crispr rna processing.the competition between viruses and hosts is played out in all branches of life. many prokaryotes have an adaptive immune system termed 'crispr' (clustered regularly interspaced short palindromic repeats) which is based on the capture of short pieces of viral dna. the captured dna is integrated into the genomic dna of the organism flanked by direct repeats, transcribed and processed to generate crrna (crispr rna) that is loaded into a variety of effector complexes. these complexes carry out sequ ...201323527601
distinctive contributions of the ribosomal p-site elements m2g966, m5c967 and the c-terminal tail of the s9 protein in the fidelity of initiation of translation in escherichia coli.the accuracy of pairing of the anticodon of the initiator trna (trna(fmet)) and the initiation codon of an mrna, in the ribosomal p-site, is crucial for determining the translational reading frame. however, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. the p-site elements, m(2)g966 (methylated by rsmd), m(5)c967 (methylated by rsmb) and the c-terminal tail of the protein s9 lie in the vicinity of trna(fmet). we investigated the role of these elements in initi ...201323530111
structures of protein-protein complexes involved in electron transfer.electron transfer reactions are essential for life because they underpin oxidative phosphorylation and photosynthesis, processes leading to the generation of atp, and are involved in many reactions of intermediary metabolism. key to these roles is the formation of transient inter-protein electron transfer complexes. the structural basis for the control of specificity between partner proteins is lacking because these weak transient complexes have remained largely intractable for crystallographic ...201323535590
specificity of processing α-glucosidase i is guided by the substrate conformation: crystallographic and in silico studies.the enzyme “glui” is key to the synthesis of critical glycoproteins in the cell.201323536181
dynamic interplay of para with the polarity protein, scy, coordinates the growth with chromosome segregation in streptomyces coelicolor.prior to bacterial cell division, the atp-dependent polymerization of the cytoskeletal protein, para, positions the newly replicated origin-proximal region of the chromosome by interacting with parb complexes assembled on pars sites located close to the origin. during the formation of unigenomic spores from multi-genomic aerial hyphae compartments of streptomyces coelicolor, para is developmentally triggered to form filaments along the hyphae; this promotes the accurate and synchronized segregat ...201323536551
amino acid substitutions in cold-adapted proteins from halorubrum lacusprofundi, an extremely halophilic microbe from antarctica.the halophilic archaeon halorubrum lacusprofundi, isolated from the perennially cold and hypersaline deep lake in antarctica, was recently sequenced and compared to 12 haloarchaea from temperate climates by comparative genomics. amino acid substitutions for 604 h. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (chogs) were determined and found to occur at 7.85% of positions invariant in proteins from mesophilic haloarchaea. the following substitutions were observed ...201323536799
the structural basis for specific decoding of aua by isoleucine trna on the ribosome.decoding of the aua isoleucine codon in bacteria and archaea requires modification of a c in the anticodon wobble position of the isoleucine trna. here, we report the crystal structure of the archaeal trna2(ile), which contains the modification agmatidine in its anticodon, in complex with the aua codon on the 70s ribosome. the structure illustrates how agmatidine confers codon specificity for aua over aug.201323542153
semiquinone and cluster n6 signals in his-tagged proton-translocating nadh:ubiquinone oxidoreductase (complex i) from escherichia coli.nadh:ubiquinone oxidoreductase (complex i) pumps protons across the membrane using downhill redox energy. the escherichia coli complex i consists of 13 different subunits named nuoa-n coded by the nuo operon. due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ~15-kb operon, purification of e. coli complex i has been technically challenging. here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoe in the op ...201323543743
inhibition of isoleucyl-trna synthetase as a potential treatment for human african trypanosomiasis.trypanosoma brucei sp. causes human african trypanosomiasis (hat; african sleeping sickness). the parasites initially proliferate in the hemolymphatic system and then invade the central nervous system, which is lethal if not treated. new drugs are needed for hat because the approved drugs are few, toxic, and difficult to administer, and drug resistance is spreading. we showed by rnai knockdown that t. brucei isoleucyl-trna synthetase is essential for the parasites in vitro and in vivo in a mouse ...201323548908
mycobacterium tuberculosis rna polymerase-binding protein a (rbpa) and its interactions with sigma factors.rna polymerase-binding protein a (rbpa), encoded by rv2050, is specific to the actinomycetes, where it is highly conserved. in the pathogen mycobacterium tuberculosis, rbpa is essential for growth and survival. rbpa binds to the β subunit of the rna polymerase where it activates transcription by unknown mechanisms, and it may also influence the response of m. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. here we report the solution structure of rbpa and identify the pr ...201323548911
ribonucleoproteins in archaeal pre-rrna processing and modification.given that ribosomes are one of the most important cellular macromolecular machines, it is not surprising that there is intensive research in ribosome biogenesis. ribosome biogenesis is a complex process. the maturation of ribosomal rnas (rrnas) requires not only the precise cleaving and folding of the pre-rrna but also extensive nucleotide modifications. at the heart of the processing and modifications of pre-rrnas in archaea and eukarya are ribonucleoprotein (rnp) machines. they are called sma ...201323554567
exploration of sitagliptin as a potential inhibitor for the m1 alanine aminopeptidase enzyme in plasmodium falciparum using computational docking.plasmodium falciparum has limited capacity for de novo amino acid synthesis and rely on degradation of host hemoglobin to maintain protein metabolism and synthesis of proteins. m1 alanine aminopeptidase enzyme of the parasite involved in the terminal degradation of host hemoglobin was subjected to in silico screening with low molecular weight protease inhibitors. the km (avg) of the enzyme m1 alanine aminopeptidase for the substrate dl - alanine β napthylamide hydrochloride was estimated as 322. ...201323559748
exclusive use of trans-editing domains prevents proline mistranslation.aminoacyl-trna synthetases (arss) catalyze the attachment of specific amino acids to cognate trnas. although the accuracy of this process is critical for overall translational fidelity, similar sizes of many amino acids provide a challenge to arss. for example, prolyl-trna synthetases (prorss) mischarge alanine and cysteine onto trna(pro). many bacterial prorss possess an alanine-specific proofreading domain (ins) but lack the capability to edit cys-trna(pro). instead, cys-trna(pro) is cleared b ...201323564458
probing the stabilizing effects of modified nucleotides in the bacterial decoding region of 16s ribosomal rna.the bacterial decoding region of 16s ribosomal rna has multiple modified nucleotides. in order to study the role of n(4),2'-o-dimethylcytidine (m(4)cm), the corresponding phosphoramidite was synthesized utilizing 5'-silyl-2'-ace chemistry. using solid-phase synthesis, m(4)cm, 5-methylcytidine (m(5)c), 3-methyluridine (m(3)u), and 2'-o-methylcytidine (cm) were site-specifically incorporated into small rnas representing the decoding regions of different bacterial species. biophysical studies were ...201323566761
combined effect of loss of the caa3 oxidase and crp regulation drives shewanella to thrive in redox-stratified environments.shewanella species are a group of facultative gram-negative microorganisms with remarkable respiration abilities that allow the use of a diverse array of terminal electron acceptors (ea). like most bacteria, s. oneidensis possesses multiple terminal oxidases, including two heme-copper oxidases (caa3- and cbb3-type) and a bd-type quinol oxidase. as aerobic respiration is energetically favored, mechanisms underlying the fact that these microorganisms thrive in redox-stratified environments remain ...201323575370
statistical evaluation of the rodin-ohno hypothesis: sense/antisense coding of ancestral class i and ii aminoacyl-trna synthetases.we tested the idea that ancestral class i and ii aminoacyl-trna synthetases arose on opposite strands of the same gene. we assembled excerpted 94-residue urgenes for class i tryptophanyl-trna synthetase (trprs) and class ii histidyl-trna synthetase (hisrs) from a diverse group of species, by identifying and catenating three blocks coding for secondary structures that position the most highly conserved, active-site residues. the codon middle-base pairing frequency was 0.35 ± 0.0002 in all-by-all ...201323576570
l1r, a27l, a33r and b5r vaccinia virus genes expressed by fowlpox recombinants as putative novel orthopoxvirus vaccines.the traditional smallpox vaccine, administered by scarification, was discontinued in the general population from 1980, because of the absence of new smallpox cases. however, the development of an effective prophylactic vaccine against smallpox is still necessary, to protect from the threat of deliberate release of the variola virus for bioterrorism and from new zoonotic infections, and to improve the safety of the traditional vaccine. preventive vaccination still remains the most effective contr ...201323578094
insight to the interaction of the dihydrolipoamide acetyltransferase (e2) core with the peripheral components in the escherichia coli pyruvate dehydrogenase complex via multifaceted structural approaches.multifaceted structural approaches were undertaken to investigate interaction of the e2 component with e3 and e1 components from the escherichia coli pyruvate dehydrogenase multienzyme complex (pdhc), as a representative of the pdhc from gram-negative bacteria. the crystal structure of e3 at 2.5 å resolution reveals similarity to other e3 structures and was an important starting point for understanding interaction surfaces between e3 and e2. biochemical studies revealed that r129e-e2 and r150e-e ...201323580650
the glove-like structure of the conserved membrane protein tatc provides insight into signal sequence recognition in twin-arginine translocation.in bacteria, two signal-sequence-dependent secretion pathways translocate proteins across the cytoplasmic membrane. although the mechanism of the ubiquitous general secretory pathway is becoming well understood, that of the twin-arginine translocation pathway, responsible for translocation of folded proteins across the bilayer, is more mysterious. tatc, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore ass ...201323583035
the yin and yang of trna: proper binding of acceptor end determines the catalytic balance of editing and aminoacylation.faithful translation of the genetic code depends on accurate coupling of amino acids with cognate transfer rnas (trnas) catalyzed by aminoacyl-trna synthetases. the fidelity of leucyl-trna synthetase (leurs) depends mainly on proofreading at the pre- and post-transfer levels. during the catalytic cycle, the trna cca-tail shuttles between the synthetic and editing domains to accomplish the aminoacylation and editing reactions. previously, we showed that the y330d mutation of escherichia coli leur ...201323585282
defining the escherichia coli seca dimer interface residues through in vivo site-specific photo-cross-linking.the motor protein seca is a core component of the bacterial general secretory (sec) pathway and is essential for cell viability. despite evidence showing that seca exists in a dynamic monomer-dimer equilibrium favoring the dimeric form in solution and in the cytoplasm, there is considerable debate as to the quaternary structural organization of the seca dimer. here, a site-directed photo-cross-linking technique was utilized to identify residues on the escherichia coli seca (ecseca) dimer interfa ...201323585536
non-complexed four cascade enzyme mixture: simple purification and synergetic co-stabilization.cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. however, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. we studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate i ...201323585905
structural basis for the enzymatic formation of the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2h)-furanone.the last step in the biosynthetic route to the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2h)-furanone (hdmf) is catalyzed by fragaria x ananassa enone oxidoreductase (faeo), earlier putatively assigned as quinone oxidoreductase (faqr). the ripening-induced enzyme catalyzes the reduction of the exocyclic double bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2h)-furanone (hmmf) in a nad(p)h-dependent manner. to elucidate the molecular mechanism of this peculia ...201323589283
life in an arsenic-containing gold mine: genome and physiology of the autotrophic arsenite-oxidizing bacterium rhizobium sp. nt-26.arsenic is widespread in the environment and its presence is a result of natural or anthropogenic activities. microbes have developed different mechanisms to deal with toxic compounds such as arsenic and this is to resist or metabolize the compound. here, we present the first reference set of genomic, transcriptomic and proteomic data of an alphaproteobacterium isolated from an arsenic-containing goldmine: rhizobium sp. nt-26. although phylogenetically related to the plant-associated bacteria, t ...201323589360
t box rna decodes both the information content and geometry of trna to affect gene expression.the t box leader sequence is an rna element that controls gene expression by binding directly to a specific trna and sensing its aminoacylation state. this interaction controls expression of amino acid-related genes in a negative feedback loop. the t box rna structure is highly conserved, but its trna binding mechanism is only partially understood. known sequence elements are the specifier sequence, which recognizes the trna anticodon, and the antiterminator bulge, which base pairs with the trna ...201323589841
crystal structure of a prokaryotic (6-4) photolyase with an fe-s cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore.the (6-4) photolyases use blue light to reverse uv-induced (6-4) photoproducts in dna. this (6-4) photorepair was thought to be restricted to eukaryotes. here we report a prokaryotic (6-4) photolyase, phrb from agrobacterium tumefaciens, and propose that (6-4) photolyases are broadly distributed in prokaryotes. the crystal structure of photolyase related protein b (phrb) at 1.45 å resolution suggests a dna binding mode different from that of the eukaryotic counterparts. a his-his-x-x-arg motif i ...201323589886
essential requirements for the detection and degradation of invaders by the haloferax volcanii crispr/cas system i-b.to fend off foreign genetic elements, prokaryotes have developed several defense systems. the most recently discovered defense system, crispr/cas, is sequence-specific, adaptive and heritable. the two central components of this system are the cas proteins and the crispr rna. the latter consists of repeat sequences that are interspersed with spacer sequences. the crispr locus is transcribed into a precursor rna that is subsequently processed into short crrnas. crispr/cas systems have been identif ...201323594992
activation and products of the cryptic secondary metabolite biosynthetic gene clusters by rifampin resistance (rpob) mutations in actinomycetes.a subset of rifampin resistance (rpob) mutations result in the overproduction of antibiotics in various actinomycetes, including streptomyces, saccharopolyspora, and amycolatopsis, with h437y and h437r rpob mutations effective most frequently. moreover, the rpob mutations markedly activate (up to 70-fold at the transcriptional level) the cryptic/silent secondary metabolite biosynthetic gene clusters of these actinomycetes, which are not activated under general stressful conditions, with the exce ...201323603745
mutations in tubg1, dync1h1, kif5c and kif2a cause malformations of cortical development and microcephaly.the genetic causes of malformations of cortical development (mcd) remain largely unknown. here we report the discovery of multiple pathogenic missense mutations in tubg1, dync1h1 and kif2a, as well as a single germline mosaic mutation in kif5c, in subjects with mcd. we found a frequent recurrence of mutations in dync1h1, implying that this gene is a major locus for unexplained mcd. we further show that the mutations in kif5c, kif2a and dync1h1 affect atp hydrolysis, productive protein folding an ...201323603762
mutations in mitochondrial ribosomal protein mrpl12 leads to growth retardation, neurological deterioration and mitochondrial translation deficiency.multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and are associated with a wide range of clinical symptoms. we report a subject, born to consanguineous parents, with growth retardation and neurological deterioration. multiple respiratory chain deficiency was found in muscle and fibroblasts of the subject as well as abnormal assembly of complexes i and iv. a microsatellite genotyping of the family members detected only one region of homozygosity on chromo ...201323603806
rna-methyltransferase trma is a dual-specific enzyme responsible for c5-methylation of uridine in both tmrna and trna.in bacteria, trans-translation rescues stalled ribosomes by the combined action of tmrna (transfer-mrna) and its associated protein smpb. the tmrna 5' and 3' ends fold into a trna-like domain (tld), which shares structural and functional similarities with trnas. as in trnas, the uuc sequence of the t-arm of the tld is post-transcriptionally modified to m (5)uψc. in trnas of gram-negative bacteria, formation of m (5)u is catalyzed by the sam-dependent methyltransferase trma, while formation of m ...201323603891
yidc occupies the lateral gate of the secyeg translocon and is sequentially displaced by a nascent membrane protein.most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved secy complex and they access the lipid phase presumably via a lateral gate in secy. in bacteria, the lipid transfer of membrane proteins from the secy channel is assisted by the secy-associated protein yidc, but details on the secy-yidc interaction are unknown. by employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the secy-yidc interface and found yidc i ...201323609445
an aromatic residue switch in enhancer-dependent bacterial rna polymerase controls transcription intermediate complex activity.the formation of the open promoter complex (rpo) in which the melted dna containing the transcription start site is located at the rna polymerase (rnap) catalytic centre is an obligatory step in the transcription of dna into rna catalyzed by rnap. in the rpo, an extensive network of interactions is established between dna, rnap and the σ-factor and the formation of functional rpo occurs via a series of transcriptional intermediates (collectively 'rpi'). a single tryptophan is ideally positioned ...201323609536
bacillus subtilis rna deprotection enzyme rpph recognizes guanosine in the second position of its substrates.the initiation of mrna degradation often requires deprotection of its 5' end. in eukaryotes, the 5'-methylguanosine (cap) structure is principally removed by the nudix family decapping enzyme dcp2, yielding a 5'-monophosphorylated rna that is a substrate for 5' exoribonucleases. in bacteria, the 5'-triphosphate group of primary transcripts is also converted to a 5' monophosphate by a nudix protein called rna pyrophosphohydrolase (rpph), allowing access to both endo- and 5' exoribonucleases. here ...201323610407
escherichia coli rimm and yjeq null strains accumulate immature 30s subunits of similar structure and protein complement.assembly of the escherichia coli 30s ribosomal subunits proceeds through multiple parallel pathways. the protein factors rimm, yjeq, rbfa, and era work in conjunction to assist at the late stages of the maturation process of the small subunit. however, it is unclear how the functional interplay between these factors occurs in the context of multiple parallel pathways. to understand how these factors work together, we have characterized the immature 30s subunits that accumulate in δrimm cells and ...201323611982
stable expression plasmids for streptomyces based on a toxin-antitoxin system.bacteria included in the genus streptomyces exhibit several attractive characteristics that make them adequate hosts for the heterologous expression of proteins. one of them is that some of its species have a high secretion capacity and hence the protein of interest could be released to the culture supernatant, facilitating downstream processing. to date, all the expression vectors described for these bacteria contain antibiotic resistance genes as selection markers. however, the use of antibiot ...201323617558
two crispr-cas systems in methanosarcina mazei strain gö1 display common processing features despite belonging to different types i and iii.the clustered regularly interspaced short palindromic repeats (crispr) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. we analyzed the two crispr-cas systems in methanosarcina mazei strain gö1. although belonging to different subtypes (i-b and iii-b), the leaders and repeats of both loci are nearly identical. also, despite many point mutations in each array, a common hairpin motif was identified in the repeats by a bioinform ...201323619576
differential regulation by ppgpp versus pppgpp in escherichia coli.both ppgpp and pppgpp are thought to function collectively as second messengers for many complex cellular responses to nutritional stress throughout biology. there are few indications that their regulatory effects might be different; however, this question has been largely unexplored for lack of an ability to experimentally manipulate the relative abundance of ppgpp and pppgpp. here, we achieve preferential accumulation of either ppgpp or pppgpp with escherichia coli strains through induction of ...201323620295
the magic spot: a ppgpp binding site on e. coli rna polymerase responsible for regulation of transcription initiation.the global regulatory nucleotide ppgpp ("magic spot") regulates transcription from a large subset of escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with rna polymerase (rnap) without binding to dna. however, ppgpp's target site on rnap, and therefore its mechanism of action, has remained unclear. we report here a binding site for ppgpp on e. coli rnap, identified by crosslinking, protease mapping, and analysis of muta ...201323623682
the mechanism of e. coli rna polymerase regulation by ppgpp is suggested by the structure of their complex.guanosine tetraphosphate (ppgpp) is an alarmone that enables bacteria to adapt to their environment. it has been known for years that ppgpp acts directly on rna polymerase (rnap) to alter the rate of transcription, but its exact target site is still under debate. here we report a crystal structure of escherichia coli rnap holoenzyme in complex with ppgpp at 4.5 å resolution. the structure reveals that ppgpp binds at an interface between the shelf and core modules on the outer surface of rnap, aw ...201323623685
bacteriophage p23-77 capsid protein structures reveal the archetype of an ancient branch from a major virus lineage.it has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. however, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. bacteriophage p23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. we have solved multiple crystal structures of the major capsid proteins vp16 and vp ...201323623731
structures of mycobacterium tuberculosis fadd10 protein reveal a new type of adenylate-forming enzyme.mycobacterium tuberculosis has a group of 34 fadd proteins that belong to the adenylate-forming superfamily. they are classified as either fatty acyl-amp ligases (faals) or fatty acyl-coa ligases based on sequence analysis. fadd10, involved in the synthesis of a virulence-related lipopeptide, was mis-annotated as a fatty acyl-coa ligase; however, it is in fact a faal that transfers fatty acids to an acyl carrier protein (rv0100). in this study, we have determined the structures of fadd10 in both ...201323625916
recognition of two distinct elements in the rna substrate by the rna-binding domain of the t. thermophilus dead box helicase hera.dead box helicases catalyze the atp-dependent destabilization of rna duplexes. whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the rna substrate. the thermus thermophilus dead-box helicase hera (for "heat-resistant rna-binding atpase") contains a c-terminal rna-binding domain (rbd). we have analyzed rna binding to the hera rbd by a combination of mutational analyses, nuclear magnetic resonance and x-r ...201323625962
identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis.the molybdenum cofactor (moco) is a redox cofactor found in all kingdoms of life, and its biosynthesis is essential for survival of many organisms, including humans. the first step of moco biosynthesis is a unique transformation of guanosine 5'-triphosphate (gtp) into cyclic pyranopterin monophosphate (cpmp). in bacteria, moaa and moac catalyze this transformation, although the specific functions of these enzymes were not fully understood. here, we report the first isolation and structural chara ...201323627491
reorganization of an intersubunit bridge induced by disparate 16s ribosomal ambiguity mutations mimics an ef-tu-bound state.after four decades of research aimed at understanding trna selection on the ribosome, the mechanism by which ribosomal ambiguity (ram) mutations promote miscoding remains unclear. here, we present two x-ray crystal structures of the thermus thermophilus 70s ribosome containing 16s rrna ram mutations, g347u and g299a. each of these mutations causes miscoding in vivo and stimulates elongation factor thermo unstable (ef-tu)-dependent gtp hydrolysis in vitro. mutation g299a is located near the inter ...201323630274
nusg-spt5 proteins-universal tools for transcription modification and communication. 201323638618
alternative excision repair pathways.alternative excision repair (aer) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of dna damage, and followed by excision of the damaged dna, repair synthesis, and ligation. the ultraviolet (uv) damage endonuclease in fungi and bacteria introduces a nick immediately 5' to various types of uv damage and initiates its excision repair that is independent of nucleotide excision repair (ner). endo iv-type apurinic/apyrimidinic (ap) endonucleases fro ...201323645854
identification of the nature of reading frame transitions observed in prokaryotic genomes.our goal was to identify evolutionary conserved frame transitions in protein coding regions and to uncover an underlying functional role of these structural aberrations. we used the ab initio frameshift prediction program, genetack, to detect reading frame transitions in 206 991 genes (fs-genes) from 1106 complete prokaryotic genomes. we grouped 102 731 fs-genes into 19 430 clusters based on sequence similarity between protein products (fs-proteins) as well as conservation of predicted position ...201323649834
tertiary structure of bacterial selenocysteine trna.selenocysteine (sec) is translationally incorporated into proteins in response to the uga codon. the trna specific to sec (trna(sec)) is first ligated with serine by seryl-trna synthetase (serrs). in the present study, we determined the 3.1 å crystal structure of the trna(sec) from the bacterium aquifex aeolicus, in complex with the heterologous serrs from the archaeon methanopyrus kandleri. the bacterial trna(sec) assumes the l-shaped structure, from which the long extra arm protrudes. although ...201323649835
affinity purification of t7 rna transcripts with homogeneous ends using aribo and crispr tags.affinity purification of rna using the aribo tag technology currently provides an ideal approach to quickly prepare rna with 3' homogeneity. here, we explored strategies to also ensure 5' homogeneity of affinity-purified rnas. first, we systematically investigated the effect of starting nucleotides on the 5' heterogeneity of a small sli rna substrate from the neurospora vs ribozyme purified from an sli-aribo precursor. a series of 32 sli rna sequences with variations in the +1 to +3 region was p ...201323657939
secretome analysis defines the major role of secdf in staphylococcus aureus virulence.the sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. the accessory sec constituent secdf has been proposed to contribute to protein export. deletion of staphylococcus aureus secdf has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. to analyse the impact of the secdf deletion in s. aureus on protein secretion, a quantitativ ...201323658837
role of loops connecting secondary structure elements in the stabilization of proteins isolated from thermophilic organisms.it has been recently discovered that the connection of secondary structure elements (ββ-unit, βα- and αβ-units) in proteins follows quite stringent principles regarding the chirality and the orientation of the structural units (koga et al., nature 2012;491:222-227). by exploiting these rules, a number of protein scaffolds endowed with a remarkable thermal stability have been designed (koga et al., nature 2012;491:222-227). by using structural databases of proteins isolated from either mesophilic ...201323661276
tetramerization reinforces the dimer interface of mnsod.two yeast manganese superoxide dismutases (mnsod), one from saccharomyces cerevisiae mitochondria (scmnsod) and the other from candida albicans cytosol (camnsodc), have most biochemical and biophysical properties in common, yet scmnsod is a tetramer and camnsodc is a dimer or "loose tetramer" in solution. although camnsodc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of camnsodc in vivo depends upon the formation of the tetram ...201323667478
aggregate-reactivation activity of the molecular chaperone clpb from ehrlichia chaffeensis.rickettsiale diseases, including human monocytic ehrlichiosis caused by ehrlichia chaffeensis, are the second leading cause of the tick-borne infections in the usa and a growing health concern. little is known about how e. chaffeensis survives the host-induced stress in vertebrate and tick hosts. a molecular chaperone clpb from several microorganisms has been reported to reactivate aggregated proteins in cooperation with the co-chaperones dnak/dnaj/grpe (kje). in this study, we performed the fir ...201323667479
a structural role for the php domain in e. coli dna polymerase iii.in addition to the core catalytic machinery, bacterial replicative dna polymerases contain a polymerase and histidinol phosphatase (php) domain whose function is not entirely understood. the php domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. in e. coli dna polymerase iii, however, the php domain has lost several metal-coordinating residues and is likely to be catalytically inactive.201323672456
peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly.the peptidoglycan (pg) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. a quantitative understanding of the relationships between pg architecture, morphogenesis, immune system activation and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell wall synthesis and cell growth. high-performance liquid chromatography (hplc) has played an important role in our ...201323679048
x-ray crystal structures of the escherichia coli rna polymerase in complex with benzoxazinorifamycins.rifampin, a semisynthetic rifamycin, is the cornerstone of current tuberculosis treatment. among many semisynthetic rifamycins, benzoxazinorifamycins have great potential for tb treatment due to their superior affinity for wild-type and rifampin-resistant mycobacterium tuberculosis rna polymerases and their reduced hepatic cyp450 induction activity. in this study, we have determined the crystal structures of the escherichia coli rna polymerase complexes with two benzoxazinorifamycins. the ansa-n ...201323679862
investigation of nadh binding, hydride transfer, and nad(+) dissociation during nadh oxidation by mitochondrial complex i using modified nicotinamide nucleotides.nadh:ubiquinone oxidoreductase (complex i) is a complicated respiratory enzyme that conserves the energy from nadh oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. during catalysis, nadh oxidation by a flavin mononucleotide is followed by electron transfer to a chain of iron-sulfur clusters. alternatively, the flavin may be reoxidized by hydrophilic electron acceptors, by artificial electron acceptors in kinetic studies, or by oxygen a ...201323683271
how to achieve high-level expression of microbial enzymes: strategies and perspectives.microbial enzymes have been used in a large number of fields, such as chemical, agricultural and biopharmaceutical industries. the enzyme production rate and yield are the main factors to consider when choosing the appropriate expression system for the production of recombinant proteins. recombinant enzymes have been expressed in bacteria (e.g., escherichia coli, bacillus and lactic acid bacteria), filamentous fungi (e.g., aspergillus) and yeasts (e.g., pichia pastoris). the favorable and very a ...201323686280
structure of the protein core of translation initiation factor 2 in apo, gtp-bound and gdp-bound forms.translation initiation factor 2 (if2) is involved in the early steps of bacterial protein synthesis. it promotes the stabilization of the initiator trna on the 30s initiation complex (ic) and triggers gtp hydrolysis upon ribosomal subunit joining. while the structure of an archaeal homologue (a/eif5b) is known, there are significant sequence and functional differences in eubacterial if2, while the trimeric eukaryotic if2 is completely unrelated. here, the crystal structure of the apo if2 protein ...201323695237
cloning, expression, purification and preliminary x-ray diffraction studies of a mycobacterial protein implicated in bacterial survival in macrophages.mycobacterium species have developed numerous strategies to avoid the antimycobacterial actions of macrophages, enabling them to survive within the generally inhospitable environment of the cell. the recently identified msmeg_5817 protein from m. smegmatis is highly conserved in mycobacterium spp. and is required for bacterial survival in macrophages. here, the cloning, expression, purification and crystallization of msmeg_5817 is reported. crystals of msmeg_5817 were grown in 1.42 m li2so4, 0.1 ...201323695579
thermostable group ii intron reverse transcriptase fusion proteins and their use in cdna synthesis and next-generation rna sequencing.mobile group ii introns encode reverse transcriptases (rts) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron rna with high processivity and fidelity. although the latter properties are potentially useful for applications in cdna synthesis and next-generation rna sequencing (rna-seq), group ii intron rts have been difficult to purify free of the intron rna, and their utility as research tools has not been inv ...201323697550
cryo-em structures of the late-stage assembly intermediates of the bacterial 50s ribosomal subunit.ribosome assembly is a process fundamental for all cellular activities. the efficiency and accuracy of the subunit assembly are tightly regulated and closely monitored. in the present work, we characterized, both compositionally and structurally, a set of in vivo 50s subunit precursors (45s), isolated from a mutant bacterial strain. our qualitative mass spectrometry data indicate that l28, l16, l33, l36 and l35 are dramatically underrepresented in the 45s particles. this protein spectrum shows i ...201323700310
crystal structure of a bioactive pactamycin analog bound to the 30s ribosomal subunit.biosynthetically and chemically derived analogs of the antibiotic pactamycin and de-6-methylsalicylyl (msa)-pactamycin have attracted recent interest as potential antiprotozoal and antitumor drugs. here, we report a 3.1-å crystal structure of de-6-msa-pactamycin bound to its target site on the thermus thermophilus 30s ribosomal subunit. although de-6-msa-pactamycin lacks the msa moiety, it shares the same binding site as pactamycin and induces a displacement of nucleic acid template bound at the ...201323702293
detection, distribution and characterization of novel superoxide dismutases from yersinia enterocolitica biovar 1a.superoxide dismutases (sods) cause dismutation of superoxide radicals to hydrogen peroxide and oxygen. besides protecting the cells against oxidative damage by endogenously generated oxygen radicals, sods play an important role in intraphagocytic survival of pathogenic bacteria. the complete genome sequences of yersinia enterocolitica strains show presence of three different sod genes. however, not much is known about the types of sods present in y. enterocolitica, their characteristics and role ...201323704955
cardioprotection by s-nitrosation of a cysteine switch on mitochondrial complex i.oxidative damage from elevated production of reactive oxygen species (ros) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. the mechanism by which the increase in ros occurs is not known, and it is unclear how this increase can be prevented. a wide variety of nitric oxide donors and s-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear. here we used a mitochondria-selective s-nitrosating agent, mitosno, to ...201323708290
r3d align web server for global nucleotide to nucleotide alignments of rna 3d structures.the r3d align web server provides online access to 'rna 3d align' (r3d align), a method for producing accurate nucleotide-level structural alignments of rna 3d structures. the web server provides a streamlined and intuitive interface, input data validation and output that is more extensive and easier to read and interpret than related servers. the r3d align web server offers a unique gallery of featured alignments, providing immediate access to pre-computed alignments of large rna 3d structures, ...201323716643
structure of an a-form rna duplex obtained by degradation of 6s rna in a crystallization droplet.in the course of a crystallographic study of a 132 nt variant of aquifex aeolicus 6s rna, a crystal structure of an a-form rna duplex containing 12 base pairs was solved at a resolution of 2.6 å. in fact, the rna duplex is part of the 6s rna and was obtained by accidental but precise degradation of the 6s rna in a crystallization droplet. 6s rna degradation was confirmed by microscopic observation of crystals and gel electrophoresis of crystallization droplets. the rna oligomers obtained form re ...201323722840
common evolutionary origin for the rotor domain of rotary atpases and flagellar protein export apparatus.the v1- and f1- rotary atpases contain a rotor that rotates against a catalytic a3b3 or α3β3 stator. the rotor f(1-γ) or v1-df is composed of both anti-parallel coiled coil and globular-loop parts. the bacterial flagellar type iii export apparatus contains a v1/f1-like atpase ring structure composed of flii6 homo-hexamer and flij which adopts an anti-parallel coiled coil structure without the globular-loop part. here we report that flij of salmonella enterica serovar typhimurium shows a rotor li ...201323724081
a new genomic tool, ultra-frequently cleaving taqii/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse dna libraries.genomics and metagenomics are currently leading research areas, with dna sequences accumulating at an exponential rate. although enormous advances in dna sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. a number of dna fragmentation methods, such as hydrodynamic sharing, sonication or dnase i fragmentation, have various drawbacks, including dna damage, poor fragmentation control, irrepr ...201323724933
role of the ribosomal p-site elements of m²g966, m⁵c967, and the s9 c-terminal tail in maintenance of the reading frame during translational elongation in escherichia coli.the ribosomal p-site hosts the peptidyl-trnas during translation elongation. which p-site elements support these trna species to maintain codon-anticodon interactions has remained unclear. we investigated the effects of p-site features of methylations of g966, c967, and the conserved c-terminal tail sequence of ser, lys, and arg (skr) of the s9 ribosomal protein in maintenance of the translational reading frame of an mrna. we generated escherichia coli strains deleted for the skr sequence in s9 ...201323729652
multiple pathways promote dynamical coupling between catalytic domains in escherichia coli prolyl-trna synthetase.aminoacyl-trna synthetases are multidomain enzymes that catalyze covalent attachment of amino acids to their cognate trna. cross-talk between functional domains is a prerequisite for this process. in this study, we investigate the molecular mechanism of site-to-site communication in escherichia coli prolyl-trna synthetase (ec prors). earlier studies have demonstrated that evolutionarily conserved and/or co-evolved residues that are engaged in correlated motion are critical for the propagation of ...201323731272
cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from plasmodium falciparum.isoprenoids are the most diverse and abundant group of natural products. in plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (fpps), turning this enzyme into a key branch point of the isoprenoid synthesis. changes in fpps activity could alter the flux of isoprenoid compounds downstream of fpps and, hence, play a central role in the regulation of a number of essential fu ...201323734739
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