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expression, purification, and characterisation of dehydroquinate synthase from pyrococcus furiosus.dehydroquinate synthase (dhqs) catalyses the second step of the shikimate pathway to aromatic compounds. dhqs from the archaeal hyperthermophile pyrococcus furiosus was insoluble when expressed in escherichia coli but was partially solubilised when kcl was included in the cell lysis buffer. a purification procedure was developed, involving lysis by sonication at 30°c followed by a heat treatment at 70°c and anion exchange chromatography. purified recombinant p. furiosus dhqs is a dimer with a su ...201121603259
hcv ires domain iib affects the configuration of coding rna in the 40s subunit's decoding groove.hepatitis c virus (hcv) uses a structured internal ribosome entry site (ires) rna to recruit the translation machinery to the viral rna and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. different ires structural domains are used in this process, which begins with direct binding of the 40s ribosomal subunit to the ires rna and involves specific manipulation of the translational machinery. we have found that upon initial 40s subunit b ...201121606179
the role of the precursor structure in the biogenesis of microrna.the human genome contains more than 1,000 microrna (mirna) genes, which are transcribed mainly by rna polymerase ii. the canonical pathway of mirna biogenesis includes the nuclear processing of primary transcripts (pri-mirnas) by the ribonuclease drosha and further cytoplasmic processing of pre-mirnas by the ribonuclease dicer. this review discusses the issue of mirna end heterogeneity generated primarily by drosha and dicer cleavage and focuses on the structural aspects of the dicer step of mir ...201121607569
cross-linking, ligation, and sequencing of hybrids reveals rna-rna interactions in yeast.many protein-protein and protein-nucleic acid interactions have been experimentally characterized, whereas rna-rna interactions have generally only been predicted computationally. here, we describe a high-throughput method to identify intramolecular and intermolecular rna-rna interactions experimentally by cross-linking, ligation, and sequencing of hybrids (clash). as validation, we identified 39 known target sites for box c/d modification-guide small nucleolar rnas (snornas) on the yeast pre-rr ...201121610164
crystal structure of alkyl hydroperoxidase d like protein pa0269 from pseudomonas aeruginosa: homology of the ahpd-like structural family.abstract: background: alkyl hydroperoxidase activity provides an important antioxidant defense for bacterial cells. the catalytic mechanism requires two peroxidases, ahpc and ahpd, where ahpd plays the role of an essential adaptor protein. results: the crystal structure of a putative ahpd from pseudomonas aeruginosa has been determined at 1.9 a. the protein has an all-helical fold with a chain topology similar to a known ahpd from mycobacterium tuberculosis despite a low overall sequence identit ...201121615954
isoprenoid precursor biosynthesis offers potential targets for drug discovery against diseases caused by apicomplexan parasites.two, simple, c5 compounds, dimethylally diphosphate and isopentenyl diphosphate, are the universal precursors of isoprenoids, a large family of natural products involved in numerous important biological processes. two distinct biosynthetic pathways have evolved to supply these precursors. humans use the mevalonate route whilst many species of bacteria including important pathogens, plant chloroplasts and apicomplexan parasites exploit the non-mevalonate pathway. the absence from humans, combined ...201121619509
allosteric nucleotide-binding site in the mitochondrial nadh:ubiquinone oxidoreductase (respiratory complex i).the rotenone-insensitive nadh:hexaammineruthenium iii (har) oxidoreductase reactions catalyzed by bovine heart and yarrowia lipolytica submitochondrial particles or purified bovine complex i are stimulated by atp and other purine nucleotides. the soluble fraction of mammalian complex i (fp) and prokaryotic complex i homolog ndh-1 in paracoccus denitrificans plasma membrane lack stimulation of their activities by atp. the stimulation appears as a decrease in apparent k(m) values for nadh and har. ...201121624365
an extensive network of information flow through the b1b/c intersubunit bridge of the yeast ribosome.yeast ribosomal proteins l11 and s18 form a dynamic intersubunit interaction called the b1b/c bridge. recent high resolution images of the ribosome have enabled targeting of specific residues in this bridge to address how distantly separated regions within the large and small subunits of the ribosome communicate with each other. mutations were generated in the l11 side of the b1b/c bridge with a particular focus on disrupting the opposing charge motifs that have previously been proposed to be in ...201121625514
crystal structure of the heme d1 biosynthesis enzyme nire in complex with its substrate reveals new insights into the catalytic mechanism of s-adenosyl-l-methionine dependent uroporphyrinogen iii methyltransferases.during the biosynthesis of heme d(1), the essential cofactor of cytochrome cd(1) nitrite reductase, the nire protein catalyzes the methylation of uroporphyrinogen iii to precorrin-2 using s-adenosyl-l-methionine (sam) as the methyl group donor. the crystal structure of pseudomonas aeruginosa nire in complex with its substrate uroporphyrinogen iii and the reaction by-product s-adenosyl-l-homocysteine (sah) was solved to 2.0 angstrom resolution. this represents the first enzyme-substrate complex s ...201121632530
the yeast rapid trna decay pathway primarily monitors the structural integrity of the acceptor and t-stems of mature trna.trnas, like other rnas, are subject to quality control steps during and after biosynthesis. we previously described a rapid trna degradation (rtd) pathway in which the 5'-3' exonucleases rat1 and xrn1 degrade mature trna(val(aac)) in yeast mutants lacking m(7)g and m(5)c, and mature trna(ser(cga)) in mutants lacking um and ac(4)c. to understand how the rtd pathway selects substrate trnas among different trnas lacking the same modifications, we used a genetic screen to examine trna(ser(cga)) vari ...201121632824
crystallization and preliminary x-ray analysis of argininosuccinate lyase from streptococcus mutans.argininosuccinate lyase (asl) is an important enzyme in arginine synthesis and the urea cycle, which are highly conserved from bacteria to eukaryotes. the gene encoding streptococcus mutans asl (smasl) was amplified and cloned into expression vector pet28a. the recombinant smasl protein was expressed in a soluble form in escherichia coli strain bl21 (de3) and purified to homogeneity by two-step column chromatography. crystals suitable for x-ray analysis were obtained and x-ray diffraction data w ...201121636911
crystallization and preliminary x-ray crystallographic analysis of dihydrouridine synthase from thermus thermophilus and its complex with trna.dihydrouridine synthase (dus) is responsible for catalyzing dihydrouridine formation in rna by the reduction of uridine. to elucidate its rna-recognition mechanism, dus from thermus thermophilus (tthdus) and its complex with trna were crystallized. diffraction data sets were collected from crystals of native and selenomethionine-substituted tthdus to resolutions of 1.70 and 2.30 å, respectively. these crystals belonged to space group p1. preliminary x-ray crystallographic analysis showed that tw ...201121636912
expression, crystallization and preliminary x-ray analysis of a ferric binding protein from thermus thermophilus hb8.a ferric binding protein from thermus thermophilus hb8 (ttfbpa) was expressed, purified and crystallized using the hanging-drop vapour-diffusion method. four different crystal forms were obtained and characterized by x-ray diffraction. two crystal forms with ttfbpa in the apo state belonged to the orthorhombic space group p2(1)2(1)2(1) (unit-cell parameters a = 42.1, b = 139.3, c = 326.5 å and a = 42.1, b = 139.3, c = 218.9 å). the third form with ttfbpa also in the apo state belonged to the mon ...201121636922
allosteric communication between the nucleotide binding domains of caseinolytic peptidase b(clpb).clpb is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the hsp70/dnak chaperone system. each of the identical protein monomer contains two nucleotide binding domains (nbd), whose atpase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. however, how communication between these sites occurs is at present poorly understood. we have studied herein the affinity of each of the nbds for nucleotides in wt ...201121642426
a new family of deamination repair enzymes in the uracil dna glycosylase superfamily.dna glycoyslases play a major role in the repair of deaminated dna damage. previous investigations identified five families within the uracil dna glycosylase (udg) superfamily. all enzymes within the superfamily studied thus far exhibit uracil dna glycosylase activity. here we identify a new class of dna glycosylases in the udg superfamily that lacks udg activity. instead, these enzymes act as hypoxanthine dna glycosylases in vitro and in vivo. molecular modeling and structure-guided mutational ...201121642431
uracil-dna glycosylase of thermoplasma acidophilumdirects long-patch base excision repair, which is promoted by deoxynucleoside triphosphates and atp/adp, into short-patch repair.hydrolytic deamination of cytosine to uracil in dna is increased in organisms adapted to high temperatures. hitherto, the uracil base excision repair (ber) pathway has only been described in two archaeons, the crenarchaeon pyrobaculum aerophilumand the euryarchaeon archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. in the former the apurinic/apyrimidinic (ap) site intermediate is removed by the sequential action of a 5'-acting ap endonuclease and a 5'-deox ...201121665970
a sulfite respiration pathway from thermus thermophilus and the key role of newly identified cytochrome côéàôéàôéç.sulfite, produced for instance during amino acid metabolism, is a very reactive and toxic compound. various detoxification mechanisms exist, but sulfite oxidoreductases (sors) are one of the major actors in sulfite remediation in bacteria and animals. here we describe the existence of an operon in the extreme thermophilic bacterium thermus thermophilus hb8 encoding both a sor and a diheme c-type cytochrome. the in vitro analysis clearly showed that the newly identified cytochrome côéàôéàôéç acts ...201121665981
high tolerance to mutations in a chlamydia trachomatis peptide deformylase loop.to determine if and how a loop region in the peptide deformylase (pdf) of chlamydia trachomatis regulates enzyme function.201121666811
the rpb2 flap loop of human rna polymerase ii is dispensable for transcription initiation and elongation.the flap domain of multisubunit rna polymerases (rnaps), also called the wall, forms one side of the rna exit channel. in bacterial rnap, the mobile part of the flap is called the flap tip and makes essential contacts with initiation and elongation factors. cocrystal structures suggest that the orthologous part of eukaryotic rnapii, called the flap loop, contacts transcription factor iib (tfiib), but the function of the flap loop has not been assessed. we constructed and tested a deletion of the ...201121670157
structural and kinetic mapping of side-chain exposure onto the protein energy landscape.identification and characterization of structural fluctuations that occur under native conditions is crucial for understanding protein folding and function, but such fluctuations are often rare and transient, making them difficult to study. native-state hydrogen exchange (nshx) has been a powerful tool for identifying such rarely populated conformations, but it generally reveals no information about the placement of these species along the folding reaction coordinate or the barriers separating t ...201121670244
carotenoid ╬▓-ring hydroxylase and ketolase from marine bacteria-promiscuous enzymes for synthesizing functional xanthophylls.marine bacteria belonging to genera paracoccus and brevundimonas of the ╬▒-proteobacteria class can produce côéäôéç-type dicyclic carotenoids containing two ╬▓-end groups (╬▓ rings) that are modified with keto and hydroxyl groups. these bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid ╬▓-ring 4(4')-ketolase (4(4')-oxygenase; crtw) and hydroxylated by carotenoid ╬▓-ring 3(3')-hydroxylase (crtz). in addition, the genus brevundimonas possesses a g ...201121673887
high resolution structure of the ba3 cytochrome c oxidase from thermus thermophilus in a lipidic environment.the fundamental chemistry underpinning aerobic life on earth involves reduction of dioxygen to water with concomitant proton translocation. this process is catalyzed by members of the heme-copper oxidase (hco) superfamily. despite the availability of crystal structures for all types of hco, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial h(+) and e(-) transport are coupled. toward addressing this problem, we report wild type and a120f mutant structu ...201121814577
gln-trnagln synthesis in a dynamic transamidosome from helicobacter pylori, where glurs2 hydrolyzes excess glu-trnagln.in many bacteria and archaea, an ancestral pathway is used where asparagine and glutamine are formed from their acidic precursors while covalently linked to trna(asn) and trna(gln), respectively. stable complexes formed by the enzymes of these indirect trna aminoacylation pathways are found in several thermophilic organisms, and are called transamidosomes. we describe here a transamidosome forming gln-trna(gln) in helicobacter pylori, an ε-proteobacterium pathogenic for humans; this transamidoso ...201121813455
interaction of mycobacterium tuberculosis elongation factor tu with gtp is regulated by phosphorylation.during protein synthesis, translation elongation factor tu (ef-tu) is responsible for the selection and binding of the cognate aminoacyl-trna to the acceptor site on the ribosome. the activity of ef-tu is dependent on its interaction with gtp. post-translational modifications such as phosphorylation are known to regulate the activity of ef-tu in several prokaryotes. although, a study on mycobacterium tuberculosis phosphoproteome showed ef-tu to be phosphorylated, the role of phosphorylation in r ...201121803988
overexpression, crystallization, and preliminary x-ray crystallographic analysis of shikimate dehydrogenase from thermotoga maritima.shikimate dehydrogenase (sdh), which catalyses the nadph-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. previous structural studies showed that sdh exists in two conformations, an open form and a closed form, and it is believed that the conformational state is crucial to understanding a catalytic mechanism. to facilitate further structural comparisons among sdhs, structural analy ...201121795804
crystallization and preliminary x-ray analysis of 4-diphosphocytidyl-2-c-methyl-d-erythritol kinase (ispe) from mycobacterium tuberculosis.the 4-diphosphocytidyl-2-c-methyl-d-erythritol kinase (ispe) from mycobacterium tuberculosis, an enzyme from the 2-c-methyl-d-erythritol 4-phosphate (mep) pathway, is crucial and essential for the survival of this pathogenic bacterium. ispe catalyzes the conversion of 4-diphosphocytidyl-2-c-methyl-d-erythritol (cdp-me) to 4-diphosphocytidyl-2-c-methyl-d-erythritol 2-phosphate (cdp-me2p) in an atp-dependent manner. solving the crystal structure of m. tuberculosis ispe will shed light on its struc ...201121795803
expression, purification, crystallization and preliminary x-ray crystallographic analysis of the hyperthermophilic nucleotidyltransferase ttha1015 from thermus thermophilus hb8.the ttha1015 gene from thermus thermophilus hb8 encodes a hyperthermophilic nucleotidyltransferase. ttha1015 has high homology to proteins belonging to two related families: the nucleotidyltransferase-domain superfamily and the dna polymerase ß-like family. however, no crystal structures of these proteins have been reported. determination of the crystal structure of ttha1015 will help in elucidation of its function and will be useful for understanding the relationship between the structure and t ...201121795793
the role of smpb and the ribosomal decoding center in licensing tmrna entry into stalled ribosomes.in bacteria, stalled ribosomes are recycled by a hybrid transfer-messenger rna (tmrna). like trna, tmrna is aminoacylated with alanine and is delivered to the ribosome by ef-tu, where it reacts with the growing polypeptide chain. tmrna entry into stalled ribosomes poses a challenge to our understanding of ribosome function because it occurs in the absence of a codon-anticodon interaction. instead, tmrna entry is licensed by the binding of its protein partner, smpb, to the ribosomal decoding cent ...201121795410
catalytic and non-catalytic roles for the mono-adp-ribosyltransferase arr in the mycobacterial dna damage response.recent evidence indicates that the mycobacterial response to dna double strand breaks (dsbs) differs substantially from previously characterized bacteria. these differences include the use of three dsb repair pathways (hr, nhej, ssa), and the card pathway, which integrates dna damage with transcription. here we identify a role for the mono-adp-ribosyltransferase arr in the mycobacterial dna damage response. arr is transcriptionally induced following dna damage and cellular stress. although arr i ...201121789183
sulfolobus mutants, generated via pcr products, which lack putative enzymes of uv photoproduct repair.in order to determine the biological relevance of two s. acidocaldarius proteins to the repair of uv photoproducts, the corresponding genes (saci_1227 and saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. the disruption used integration by homologous recombination of a functional but heterologous pyre gene, promoted by short sequences attached to both ends via pcr. the phenotypic analyses of the disruptants confirmed that orf saci_122 ...201121785574
identification and characterization of a re-citrate synthase in dehalococcoides strain cbdb1.the genome annotations of all sequenced dehalococcoides strains lack a citrate synthase although physiological experiments have indicated that such an activity should be encoded. we here report that a re-face specific citrate synthase is synthesized by dehalococcoides strain cbdb1 and that this function is encoded by the gene cbdba1708 (ncbi accession number cai83711), previously annotated as homocitrate synthase. gene cbdba1708 was heterologously expressed in e. coli and the recombinant enzyme ...201121784924
structural and biochemical analysis of the nuclease domain of the clustered regularly interspaced short palindromic repeat (crispr) associated protein 3(cas3).rna transcribed from clustered regularly interspaced short palindromic repeats (crisprs) protects many prokaryotes from invasion by foreign dna such as viruses, conjugative plasmids and transposable elements. crispr-associated protein 3 (cas3) is essential for this crispr protection and is thought to mediate cleavage of the foreign dna through its n-terminal histidine-aspartate (hd) domain. we report here the 1.8 å crystal structure of the hd domain of cas3 from thermus thermophilus hb8. structu ...201121775431
role of trna amino acid-accepting end in aminoacylation and its quality control.aminoacyl-trna synthetases (aarss) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and trna molecules. the greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. to reduce mischarging of trnas by non-cognate amino acids, aarss have evolved an editing activity in a second active site to cleave the incorrect aminoacyl-trnas. editing occurs after translocation of the aminoacyl-cca(76) end t ...201121775341
molecular basis for the selectivity of antituberculosis compounds capreomycin and viomycin.capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics which are particularly active against mycobacterium tuberculosis, including multi-drug resistant strains. both antibiotics bind across the ribosomal interface involving 23s rrna helix 69 (h69) and 16s rrna helix 44 (h44). the binding site of tuberactinomycins in h44 partially overlaps with that of aminoglycosides and they share with these drugs the side effect of irreversible hearing loss. here we studied t ...201121768509
diverse substrate recognition and hydrolysis mechanisms of human nudt5.human nudt5 (hnudt5) hydrolyzes various modified nucleoside diphosphates including 8-oxo-dgdp, 8-oxo-dadp and adp-ribose (adpr). however, the structural basis of the broad substrate specificity remains unknown. here, we report the crystal structures of hnudt5 complexed with 8-oxo-dgdp and 8-oxo-dadp. these structures reveal an unusually different substrate-binding mode. in particular, the positions of two phosphates (a and ß phosphates) of substrate in the 8-oxo-dgdp and 8-oxo-dadp complexes are ...201121768126
human fatty acid transport protein 2a/very long chain acyl coa synthetase 1 (fatp2a/acsvl1) has a preference in mediating the channeling of exogenous n-3 fatty acids into phosphatidylinositol.the trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to coa thioesters. members of the fatty acid transport protein/very long chain acyl coa synthetase (fatp/acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. we have expressed two naturally occurring splice variants of fatp2 (acsvl1) in yeast and 293t-rex cells and addressed their roles in f ...201121768100
redox-coupled protonation of respiratory complex i: the hydrophilic domain.respiratory complex i, nadh:ubiquinone oxidoreductase, is a large and complex integral membrane enzyme found in respiring bacteria and mitochondria. it is responsible in part for generating the proton gradient necessary for atp production. complex i serves as both a proton pump and an entry point for electrons into the respiratory chain. although complex i is one of the most important of the respiratory complexes, it is also one of the least understood, with detailed structural information only ...201121767496
staphylococcus lugdunensis isdg liberates iron from host heme.staphylococcus lugdunensis is often found as part of the normal flora of human skin but has the potential to cause serious infections even in healthy individuals. it remains unclear what factors enable s. lugdunensis to transition from a skin commensal to an invasive pathogen. analysis of the complete genome reveals a putative iron-regulated surface determinant (isd) system encoded within s. lugdunensis. in other bacteria the isd system permits the utilization of host heme as a source of nutrien ...201121764939
a transcript cleavage factor of mycobacterium tuberculosis important for its survival.after initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the rna polymerase (rnap). gre factors are one such group conserved across bacteria. they regulate transcription by projecting their n-terminal coiled-coil domain into the active center of rnap through the secondary channel and stimulating hydrolysis of the newly synthesized rna in backtracked elongation complexes. rv1080c is a putative gre factor (mtbgre) in the geno ...201121760927
probing the protonation/deprotonation of tyrosine residues in cytochrome ba3 oxidase from thermus thermophilus by time-resolved step-scan ftir spectroscopy.elucidating the properties of the heme fe-cu(b) binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme, but also the events related with the release of the produced water molecules. the time-resolved step-scan ftir difference spectra show the ?7a(co) of the protonated form of y residues at 1247 cm(-1) and that of the deprotonated form at 1301 cm(-1). by monitoring the intensity ...201121757723
characterization of the deoxynucleotide triphosphate triphosphohydrolase (dntpase) activity of the ef1143 protein from enterococcus faecalis and crystal structure of the activator-substrate complex.the ef1143 protein from enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dntpases) from escherichia coli and thermus thermophilus. these dntpases are important components in the regulation of the dntp pool in bacteria. biochemical assays of the ef1143 dntpase activity demonstrated nonspecific hydrolysis of all canonical dntps in the presence of mn2+. in contrast, with mg2+ hydrolysis required the presence of dgtp as an effector, activating the degr ...201121757692
leaderless genes in bacteria: clue to the evolution of translation initiation mechanisms in prokaryotes.abstract: background: shine-dalgarno (sd) signal has long been viewed as the dominant translation initiation signal in prokaryotes. recently, leaderless genes, which lack 5'-untranslated regions (5'-utr) on their mrnas, have been shown abundant in archaea. however, current large-scale in silico analyses on initiation mechanisms in bacteria are mainly based on the sd-led initiation way, other than the leaderless one. the study of leaderless genes in bacteria remains open, which causes uncertain u ...201121749696
characterization of the mitochondrial atp synthase from yeast saccharomyces cerevisae.the mitochondrial atp synthase from yeast s. cerevisiae has been genetically modified, purified in a functional form, and characterized with regard to lipid requirement, compatibility with a variety of detergents, and the steric limit with rotation of the central stalk has been assessed. the atp synthase has been modified on the n-terminus of the ß-subunit to include a his(6) tag for ni-chelate affinity purification. the enzyme is purified by a two-step procedure from submitochondrial particles ...201121748405
expression, purification and preliminary crystallographic analysis of o-acetylhomoserine sulfhydrylase from mycobacterium tuberculosis.the gene product of the open reading frame rv3340 from mycobacterium tuberculosis is annotated as encoding a probable o-acetylhomoserine (oah) sulfhydrylase (metc), an enzyme that catalyzes the last step in the biosynthesis of methionine, which is an essential amino acid in bacteria and plants. following overexpression in escherichia coli, the m. tuberculosis metc enzyme was purified and crystallized using the hanging-drop vapor-diffusion method. native diffraction data were collected from cryst ...201121821905
community ecology of hot spring cyanobacterial mats: predominant populations and their functional potential.phototrophic microbial mat communities from 60-¦c and 65-¦c regions in the effluent channels of mushroom and octopus springs (yellowstone national park, wy, usa) were investigated by shotgun metagenomic sequencing. analyses of assembled metagenomic sequences resolved six dominant chlorophototrophic populations and permitted the discovery and characterization of undescribed but predominant community members and their physiological potential. linkage of phylogenetic marker genes and functional gen ...201121697961
kinetic and chemical mechanisms of homocitrate synthase from thermus thermophilus.the homocitrate synthase from thermus thermophilus (tthcs) is a metal-activated enzyme with either mg(2+) or mn(2+) capable of serving as the divalent cation. the enzyme exhibits a sequential kinetic mechanism. the mechanism is steady state ordered with +¦-ketoglutarate (+¦-kg) binding prior to acetyl-coa (accoa) with mn(2+), whereas it is steady state random with mg(2+), suggesting a difference in the competence of the e-àmn-à+¦-kg-àaccoa and e-àmg-à+¦-kg-àaccoa complexes. the mechanism is supp ...201121733842
mechanisms and evolution of oxidative sulfur metabolism in green sulfur bacteria.green sulfur bacteria (gsb) constitute a closely related group of photoautotrophic and thiotrophic bacteria with limited phenotypic variation. they typically oxidize sulfide and thiosulfate to sulfate with sulfur globules as an intermediate. based on genome sequence information from 15 strains, the distribution and phylogeny of enzymes involved in their oxidative sulfur metabolism was investigated. at least one homolog of sulfide:quinone oxidoreductase (sqr) is present in all strains. in all sul ...201121833341
a single methyltransferase yefa (rlmcd) catalyses both m5u747 and m5u1939 modifications in bacillus subtilis 23s rrna.methyltransferases that use s-adenosylmethionine (adomet) as a cofactor to catalyse 5-methyl uridine (m(5)u) formation in trnas and rrnas are widespread in bacteria and eukaryota, and are also found in certain archaea. these enzymes belong to the cog2265 cluster, and the gram-negative bacterium escherichia coli possesses three paralogues. these comprise the methyltransferases trma that targets u54 in trnas, rlmc that modifies u747 in 23s rrna and rlmd that is specific for u1939 in 23s rrna. the ...201121824914
dynamics of gene duplication in the genomes of chlorophyll d-producing cyanobacteria: implications for the ecological niche.gene duplication may be an important mechanism for the evolution of new functions and for the adaptive modulation of gene expression via dosage effects. here, we analyzed the fate of gene duplicates for two strains of a novel group of cyanobacteria (genus acaryochloris) that produces the far-red light absorbing chlorophyll d as its main photosynthetic pigment. the genomes of both strains contain an unusually high number of gene duplicates for bacteria. as has been observed for eukaryotic genomes ...201121697100
structural classification and properties of ketoacyl synthases.ketoacyl synthases (kss) catalyze condensing reactions combining acyl-coa or acyl-acyl carrier protein (acp) with malonyl-coa to form 3-ketoacyl-coa, or with malonyl-acp to form 3-ketoacyl-acp. in each case the resulting acyl chain is two carbon atoms longer than before, and co(2) and either coa or acp are formed. kss also join other activated molecules in the polyketide synthesis cycle. our classification of kss by their primary and tertiary structures instead of by their substrates and the rea ...201121830247
glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass.industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. compost-derived microbial consortia were adapted to switchgrass at 60-¦c to develop thermophilic biomass-degrading consortia for detailed studies. microbial community analysis using small-subunit rrna gene am ...201121724886
rna transcript 3'-proximal sequence affects translocation bias of rna polymerase.translocation of rna polymerase on dna is thought to involve oscillations between pretranslocated and posttranslocated states that are rectified by nucleotide addition or pyrophosphorolysis. the pretranslocated register is also a precursor to transcriptional pause states that mediate regulation of transcript elongation. however, the determinants of bias between the pretranslocated and posttranslocated states are incompletely understood. to investigate translocation bias in multisubunit rna polym ...201121739957
kinetic design of the respiratory oxidases.energy conservation in all kingdoms of life involves electron transfer, through a number of membrane-bound proteins, associated with proton transfer across the membrane. in aerobic organisms, the last component of this electron-transfer chain is a respiratory heme-copper oxidase that catalyzes reduction of o(2) to h(2)o, linking this process to transmembrane proton pumping. so far, the molecular mechanism of proton pumping is not known for any system that is driven by electron transfer. here, we ...201121690359
engineering the respiratory complex i to an energy-converting nadph:ubiquinone oxidoreductase.the respiratory complex i couples the electron transfer from nadh to ubiquinone with a translocation of protons across the membrane. its nucleotide binding site is made up of a unique rossmann fold to accommodate the binding of the substrate nadh and of the primary electron acceptor flavin mononucleotide. binding of nadh includes interactions of the hydroxyl groups of the adenosine ribose with a conserved glutamic acid residue. structural analysis revealed that due to steric hinderance and elect ...201121832062
rnase h-dependent pcr (rhpcr): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers.abstract: background: the polymerase chain reaction (pcr) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. while high specificity is often achieved, biological requirements sometimes require that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. results: pyrococcus abyssi (p.a.) rnase h2 was used to enable pcr to be performed using blocke ...201121831278
the elusive middle domain of hsp104 and clpb: location and function.hsp104 in yeast and clpb in bacteria are homologous, hexameric aaa+ proteins and hsp100 chaperones, which function in the stress response as ring-translocases that drive protein disaggregation and reactivation. both hsp104 and clpb contain a distinctive coiled-coil middle domain (md) inserted in the first aaa+ domain, which distinguishes them from other aaa+ proteins and hsp100 family members. here, we focus on recent developments concerning the location and function of the md in these hexameric ...201121843558
the elusive middle domain of hsp104 and clpb: location and function.hsp104 in yeast and clpb in bacteria are homologous, hexameric aaa+ proteins and hsp100 chaperones, which function in the stress response as ring-translocases that drive protein disaggregation and reactivation. both hsp104 and clpb contain a distinctive coiled-coil middle domain (md) inserted in the first aaa+ domain, which distinguishes them from other aaa+ proteins and hsp100 family members. here, we focus on recent developments concerning the location and function of the md in these hexameric ...201121843558
phylogenetic sequence variations in bacterial rrna affect species-specific susceptibility to drugs targeting protein synthesis.antibiotics targeting the bacterial ribosome typically bind to highly conserved rrna regions with only minor phylogenetic sequence variations. it is unclear whether these sequence variations affect antibiotic susceptibility or resistance development. to address this question, we have investigated the drug binding pockets of aminoglycosides and macrolides/ketolides. the binding site of aminoglycosides is located within helix 44 of the 16s rrna (a site); macrolides/ketolides bind to domain v of th ...201121730122
towards a rigorous network of protein-protein interactions of the model sulfate reducer desulfovibrio vulgaris hildenborough.protein-protein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. the aforementioned tools have been extensively applied to study escherichia coli and other aerobes and more recently to study the stress response behavior of desulfovibrio vulgaris hildenborough, a model obligate anaerobe and sulfate reducer and the subject of this study. here we carried out affinity purification follow ...201121738675
alterations in the {beta} flap and {beta}' dock domains of the rna polymerase abolish nusa-mediated feedback regulation of the mety-nusa-infb operon.the rimm protein in escherichia coli is important for the in vivo maturation of 30s ribosomal subunits and a +örimm mutant grows poorly due to assembly and translational defects. these deficiencies are suppressed partially by mutations that increase the synthesis of another assembly protein, rbfa, encoded by the mety-nusa-infb operon. among these suppressors are mutations in nusa that impair the nusa-mediated negative-feedback regulation at internal intrinsic transcriptional terminators of the m ...201121685293
unveiling the structural basis for translational ambiguity tolerance in a human fungal pathogen.in a restricted group of opportunistic fungal pathogens the universal leucine cug codon is translated both as serine (97%) and leucine (3%), challenging the concept that translational ambiguity has a negative impact in living organisms. to elucidate the molecular mechanisms underlying the in vivo tolerance to a nonconserved genetic code alteration, we have undertaken an extensive structural analysis of proteins containing cug-encoded residues and solved the crystal structures of the two natural ...201121825144
difference in the distribution pattern of substrate enzymes in the metabolic network of escherichia coli, according to chaperonin requirement.abstract:201121702926
allosteric drugs: the interaction of antitumor compound mkt-077 with human hsp70 chaperones.hsp70 (heat shock protein 70-ákda) chaperones are key to cellular protein homeostasis. however, they also have the ability to inhibit tumor apoptosis and contribute to aberrant accumulation of hyperphosphorylated tau in neuronal cells affected by tauopathies, including alzheimer's disease. hence, hsp70 chaperones are increasingly becoming identified as targets for therapeutic intervention in these widely abundant diseases. hsp70 proteins are allosteric machines and offer, besides classical activ ...201121708173
expression, purification, crystallization and preliminary crystallographic analysis of cg1458: a novel oxaloacetate decarboxylase from corynebacterium glutamicum.oxaloacetate decarboxylase catalyses the decarboxylation of oxaloacetate to pyruvate and co(2). recently, the corynebacterium glutamicum gene product cg1458 was determined to be a soluble oxaloacetate decarboxylase. to elucidate the mechanism of oxaloacetate decarboxylation by cg1458, recombinant cg1458 was purified and crystallized. the best crystal was grown from 0.2ôçàm mgcl(2), 0.1ôçàm bis-tris ph 6.0, 25%(w/v) polyethylene glycol 3350 using the hanging-drop method. the crystals belonged to ...201121821907
mechanistic analysis of trehalose synthase from mycobacterium smegmatis.trehalose synthase (tres) catalyzes the reversible interconversion of maltose and trehalose and has been recently shown to function primarily in the mobilisation of trehalose as a glycogen precursor. consequently the mechanism of this intriguing isomerase is of both academic and potential pharmacological interest. tres catalyzes the hydrolytic cleavage of +¦-aryl glucosides, as well as +¦-glucosyl fluoride, thereby allowing facile continuous assays. reaction of tres with 5-fluoroglycosyl fluorid ...201121840994
cbpa acts as a modulator of hspr repressor dna binding activity in helicobacter pylori.the ability of pathogens to cope with disparate environmental stresses is a crucial feature for bacterial survival and for the establishment of a successful infection and colonization of the host; in this respect, chaperones and heat-shock proteins (hsps) play a fundamental role in host-pathogen interactions. in helicobacter pylori, the expression of the major hsps is tightly regulated through dedicated transcriptional repressors (named hspr and hrca), as well as via a groesl-dependent post-tran ...201121840971
substrate trna recognition mechanism of a multi-site-specific trna methyltransferase, aquifex aeolicus trm1, based on the x-ray crystal structure.archaeal and eukaryotic trna (n(2)-, n(2)-guanine) dimethyltransferase (trm1) produces n(2)-, n(2)-dimethylguanine at position 26 in trna. in contrast, trm1 from aquifex aeolicus, a hyper-thermophilic eubacterium, modifies g27 as well as g26. here, a gel-mobility shift assay revealed that the t-arm in trna is the binding site of a. aeolicus trm1. to address the multi-site specificity, we performed an x-ray crystal structure study. the overall structure of a. aeolicus trm1 is similar to that of a ...201121844194
overexpression of a cytosolic pyrophosphatase (tgppase) reveals a regulatory role of pyrophosphate in glycolysis for toxoplasma gondii.pyrophosphate (ppi) is a critical element of cellular metabolism as both an energy donor and as an allosteric regulator of several metabolic pathways. the apicomplexan parasite, toxoplasma gondii, uses ppi in place of atp as an energy donor in at least two reactions: the glycolytic ppi-dependent phosphofructokinase (pfk), and the proton translocating vacuolar pyrophosphatase (v-h+-ppase). in the present work, we report the cloning, expression, and characterization of a cytosolic pyrophosphatase ...201121831041
how are "atypical" sulfite dehydrogenases linked to cell metabolism? interactions between the sort sulfite dehydrogenase and small redox proteins.sulfite dehydrogenases (sdhs) are enzymes that catalyze the oxidation of the toxic and mutagenic compound sulfite to sulfate, thereby protecting cells from adverse effects associated with sulfite exposure. while some bacterial sdhs that have been characterized to date are able to use cytochrome c as an electron acceptor, the majority of these enzymes prefer ferricyanide as an electron acceptor and have therefore been termed "atypical" sdhs. identifying the natural electron acceptor of these enzy ...201121833314
crystallization and preliminary x-ray analysis of a cold-active +¦-galactosidase from the psychrotrophic and halotolerant planococcus sp. l4.+¦-galactosidases catalyze the hydrolysis of a galactosyl moiety from the nonreducing termini of oligosaccharides or from glycosides. a novel gh family 42 cold-active +¦-galactosidase identified from the psychrotrophic and halotolerant planococcus sp. l4 (bgap) was crystallized and a complete data set was collected from a single frozen crystal on an in-house x-ray source. the crystal diffracted to 2.8ôçà+à resolution and belonged to space group p1, with unit-cell parameters a = 104.29, b = 118.1 ...201121821893
sirna repositioning for guide strand selection by human dicer complexes.the human ribonuclease dicer and its double-stranded rna (dsrna)-binding protein (dsrbp) partners trbp and pact play important roles in the biogenesis of regulatory rnas. following dicing, one dsrna product strand is preferentially assembled into an rna-induced silencing complex (risc). the mechanism of strand selection in humans and the possible role of dicer in this process remain unclear. here we demonstrate that dsrnas undergo significant repositioning within dicer complexes following dicing ...201121726814
formation of m2g6 in methanocaldococcus jannaschii trna catalyzed by the novel methyltransferase trm14.the modified nucleosides n(2)-methylguanosine and -dimethylguanosine in transfer rna occur at five positions in the d and anticodon arms, and at positions g6 and g7 in the acceptor stem. trm1 and trm11 enzymes are known to be responsible for several of the d/anticodon arm modifications, but methylases catalyzing post-transcriptional m(2)g synthesis in the acceptor stem are uncharacterized. here, we report that the mj0438 gene from methanocaldococcus jannaschii encodes a novel s-adenosylmethionin ...201121693558
generation of targeted deletions in the genome of rhodothermus marinus.the aim of this work was to develop an approach for chromosomal engineering of the thermophile rhodothermus marinus. a selection strategy for r. marinus had previously been developed; this strategy was based on complementing a restriction-negative trpb strain with the r. marinus trpb gene. the current work identified an additional selective marker, pura, which encodes adenylosuccinate synthase and confers adenine prototrophy. in a two-step procedure, the available trp(+) selection was used durin ...201121705543
vma8p-gfp fusions can be functionally incorporated into v-atpase, suggesting structural flexibility at the top of v1.the vacuolar atpase (v-atpase) complex of yeast (saccharomyces cerevisiae) is comprised of two sectors, v(1) (catalytic) and v(o) (proton transfer). the hexameric (a(3)b(3)) cylinder of v(1) has a central cavity that must accommodate at least part of the rotary stalk of v-atpase, a key component of which is subunit d (vma8p). recent electron microscopy (em) data for the prokaryote v-atpase complex (thermus thermophilus) suggest that subunit d penetrates deeply into the central cavity. the functi ...201121845105
the dgt gene of escherichia coli facilitates thymine utilization in thymine-requiring strains.the escherichia coli dgtp triphosphohydrolase (dgtpase) encoded by the dgt gene catalyses the hydrolysis of dgtp to deoxyguanosine and triphosphate. the recent discovery of a mutator effect associated with deletion of dgt indicated participation of the triphosphohydrolase in preventing mutagenesis. here, we have investigated the possible involvement of dgt in facilitating thymine utilization through its ability to provide intracellular deoxyguanosine, which is readily converted by the deod phosp ...201121736641
phenylacetyl coenzyme a is an effector molecule of the tetr family transcriptional repressor paar from thermus thermophilushb8.phenylacetic acid (paa) is a common intermediate in the catabolic pathways of several structurally related aromatic compounds. it is converted into phenylacetyl coenzyme a (pa-coa), which is degraded to general metabolites by a set of enzymes. within the genome of the extremely thermophilic bacterium thermus thermophilushb8, a cluster of genes, including a tetr family transcriptional regulator, may be involved in paa degradation. the gene product, which we named t. thermophiluspaar, negatively r ...201121725002
characterization of two malaria parasite organelle translation elongation factor g proteins: the likely targets of the anti-malarial fusidic acid.malaria parasites harbour two organelles with bacteria-like metabolic processes that are the targets of many anti-bacterial drugs. one such drug is fusidic acid, which inhibits the translation component elongation factor g. the response of p. falciparum to fusidic acid was characterised using extended sybr-green based drug trials. this revealed that fusidic acid kills in vitro cultured p. falciparum parasites by immediately blocking parasite development. two bacterial-type protein translation el ...201121695207
structural contribution of the c-terminal segments of nuol (nd5) and nuom (nd4) subunits of complex i from e. coli.the proton-translocating nadh-quinone oxidoreductase (complex i/ndh-1) is a multi-subunit enzymatic complex. it has a characteristic l-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. the membrane domain contains three antiporter-like subunits (nuol, nuom and nuon, escherichia coli naming) that are considered to be involved in the proton translocation. deletion of either nuol or nuom resulted in an incomplete assembly of ndh-1 and a total loss of t ...201121835926
Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18.5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular ...201121685364
The structural biology of +¦-barrel membrane proteins: a summary of recent reports.The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts all contain transmembrane +¦-barrel proteins. These +¦-barrel proteins serve essential functions in cargo transport and signaling and are also vital for membrane biogenesis. They have also been adapted to perform a diverse set of important cellular functions including acting as porins, transporters, enzymes, virulence factors and receptors. Recent structures of transmembrane +¦-barrels include that of a full length aut ...201121719274
The elusive third subunit IIa of the bacterial B-type oxidases: the enzyme from the hyperthermophile Aquifex aeolicus.The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba(3)-type two-subunit (subunits I and II) enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper ...201121738733
adaptation of aerobic respiration to low o2 environments.aerobic respiration in bacteria, archaea, and mitochondria is performed by oxygen reductase members of the heme-copper oxidoreductase superfamily. these enzymes are redox-driven proton pumps which conserve part of the free energy released from oxygen reduction to generate a proton motive force. the oxygen reductases can be divided into three main families based on evolutionary and structural analyses (a-, b- and c-families), with the b- and c-families evolving after the a-family. the a-family ut ...201121844375
the rela/spot homolog (rsh) superfamily: distribution and functional evolution of ppgpp synthetases and hydrolases across the tree of life.rela/spot homologue (rsh) proteins, named for their sequence similarity to the rela and spot enzymes of escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppgpp, activator of the "stringent" response and regulator of cellular metabolism. the classical "long" rshs rel, rela and spot with the ppgpp hydrolase, synthetase, tgs and act domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppgpp- ...201121858139
structure of nitrilotriacetate monooxygenase component b from mycobacterium thermoresistibile.mycobacterium tuberculosis belongs to a large family of soil bacteria which can degrade a remarkably broad range of organic compounds and utilize them as carbon, nitrogen and energy sources. it has been proposed that a variety of mycobacteria can subsist on alternative carbon sources during latency within an infected human host, with the help of enzymes such as nitrilotriacetate monooxygenase (nta-mo). nta-mo is a member of a class of enzymes which consist of two components: a and b. while compo ...201121904057
francisella rna polymerase contains a heterodimer of non-identical alpha subunits.abstract: background: all sequenced genomes of representatives of the francisella genus contain two rpoa genes, which encode non-identical rna polymerase (rnap) subunits, alpha1 and alpha2. in all other bacteria studied to date, a dimer of identical alpha subunits initiates the assembly of the catalytically proficient rnap core (subunit composition (alpha)2/beta/beta'). based on an observation that both alpha1 and alpha2 are incorporated into francisella rnap, charity et al. (2007) previously s ...201122108176
sequence of the hyperplastic genome of the naturally competent thermus scotoductus sa-01.many strains of thermus have been isolated from hot environments around the world. thermus scotoductus sa-01 was isolated from fissure water collected 3.2 km below surface in a south african gold mine. the isolate is capable of dissimilatory iron reduction, growth with oxygen and nitrate as terminal electron acceptors and the ability to reduce a variety of metal ions, including gold, chromate and uranium, was demonstrated. the genomes from two different thermus thermophilus strains have been com ...201122115438
the universally conserved prokaryotic gtpases.members of the large superclass of p-loop gtpases share a core domain with a conserved three-dimensional structure. in eukaryotes, these proteins are implicated in various crucial cellular processes, including translation, membrane trafficking, cell cycle progression, and membrane signaling. as targets of mutation and toxins, gtpases are involved in the pathogenesis of cancer and infectious diseases. in prokaryotes also, it is hard to overestimate the importance of gtpases in cell physiology. nu ...201121885683
the origin of a derived superkingdom: how a gram-positive bacterium crossed the desert to become an archaeon.the tree of life is usually rooted between archaea and bacteria. we have previously presented three arguments that support placing the root of the tree of life in bacteria. the data have been dismissed because those who support the canonical rooting between the prokaryotic superkingdoms cannot imagine how the vast divide between the prokaryotic superkingdoms could be crossed.201121356104
the bridge helix of rna polymerase acts as a central nanomechanical switchboard for coordinating catalysis and substrate movement.the availability of in vitro assembly systems to produce recombinant archaeal rna polymerases (rnaps) offers one of the most powerful experimental tools for investigating the still relatively poorly understood molecular mechanisms underlying rnap function. over the last few years, we pioneered new robot-based high-throughput mutagenesis approaches to study structure/function relationships within various domains surrounding the catalytic center. the bridge helix domain, which appears in numerous ...201122312317
the bridge helix of rna polymerase acts as a central nanomechanical switchboard for coordinating catalysis and substrate movement.the availability of in vitro assembly systems to produce recombinant archaeal rna polymerases (rnaps) offers one of the most powerful experimental tools for investigating the still relatively poorly understood molecular mechanisms underlying rnap function. over the last few years, we pioneered new robot-based high-throughput mutagenesis approaches to study structure/function relationships within various domains surrounding the catalytic center. the bridge helix domain, which appears in numerous ...201122312317
transcription initiation factor dksa has diverse effects on rna chain elongation.bacterial transcription factors dksa and greb belong to a family of coiled-coil proteins that bind within the secondary channel of rna polymerase (rnap). these proteins display structural homology but play different regulatory roles. dksa disrupts rnap interactions with promoter dna and inhibits formation of initiation complexes, sensitizing rrna synthesis to changes in concentrations of ppgpp and ntps. gre proteins remodel the rnap active site and facilitate cleavage of the nascent rna in elong ...201122210857
transcription initiation factor dksa has diverse effects on rna chain elongation.bacterial transcription factors dksa and greb belong to a family of coiled-coil proteins that bind within the secondary channel of rna polymerase (rnap). these proteins display structural homology but play different regulatory roles. dksa disrupts rnap interactions with promoter dna and inhibits formation of initiation complexes, sensitizing rrna synthesis to changes in concentrations of ppgpp and ntps. gre proteins remodel the rnap active site and facilitate cleavage of the nascent rna in elong ...201122210857
the dna exonucleases of escherichia coli.dna exonucleases, enzymes that hydrolyze phosphodiester bonds in dna from a free end, play important cellular roles in dna repair, genetic recombination and mutation avoidance in all organisms. this article reviews the structure, biochemistry, and biological functions of the 17 exonucleases currently identified in the bacterium escherichia coli. these include the exonucleases associated with dna polymerases i (pola), ii (polb), and iii (dnaq/mutd); exonucleases i (xona/sbcb), iii (xtha), iv, vii ...201126442508
enzymatic synthesis of long double-stranded dna labeled with haloderivatives of nucleobases in a precisely pre-determined sequence.restriction endonucleases are widely applied in recombinant dna technology. among them, enzymes of class iis, which cleave dna beyond recognition sites, are especially useful. we use bsai enzyme for the pinpoint introduction of halogen nucleobases into dna. this has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective.201121864341
genetic tool development underpins recent advances in thermophilic whole-cell biocatalysts.the environmental value of sustainably producing bioproducts from biomass is now widely appreciated, with a primary target being the economic production of fuels such as bioethanol from lignocellulose. the application of thermophilic prokaryotes is a rapidly developing niche in this field, driven by their known catabolic versatility with lignocellulose-derived carbohydrates. fundamental to the success of this work has been the development of reliable genetic and molecular systems. these technica ...201121310009
microbial ecology of the dark ocean above, at, and below the seafloor.the majority of life on earth--notably, microbial life--occurs in places that do not receive sunlight, with the habitats of the oceans being the largest of these reservoirs. sunlight penetrates only a few tens to hundreds of meters into the ocean, resulting in large-scale microbial ecosystems that function in the dark. our knowledge of microbial processes in the dark ocean-the aphotic pelagic ocean, sediments, oceanic crust, hydrothermal vents, etc.-has increased substantially in recent decades. ...201121646433
mechanism of bacterial transcription initiation: rna polymerase - promoter binding, isomerization to initiation-competent open complexes, and initiation of rna synthesis.initiation of rna synthesis from dna templates by rna polymerase (rnap) is a multi-step process, in which initial recognition of promoter dna by rnap triggers a series of conformational changes in both rnap and promoter dna. the bacterial rnap functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex dna in the active site cleft and then separating the nontemplate and template strands in the region surrounding ...201121371479
x-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides.we have determined the x-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage n4 rna polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit rna polymerase. as observed during t7 rna polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the o-helix, but only the correct base pairing between the +2 substrate and dna b ...201121321236
seleno-detergent mad phasing of leukotriene c4 synthase in complex with dodecyl-β-d-selenomaltoside.dodecyl-β-d-selenomaltoside (seddm) is a seleno-detergent with a β-glycosidic seleno-ether in place of the ether moiety in dodecyl-β-d-maltoside. seleno-detergents are candidates for heavy-atom agents in experimental phasing of membrane proteins in protein crystallography. crystals of a nuclear membrane-embedded enzyme, leukotriene c(4) synthase (ltc(4)s), in complex with seddm were prepared and a multiwavelength anomalous diffraction (mad) experiment was performed. the seddm in the ltc(4)s crys ...201122139193
ucsf chimera, modeller, and imp: an integrated modeling system.structural modeling of macromolecular complexes greatly benefits from interactive visualization capabilities. here we present the integration of several modeling tools into ucsf chimera. these include comparative modeling by modeller, simultaneous fitting of multiple components into electron microscopy density maps by imp multifit, computing of small-angle x-ray scattering profiles and fitting of the corresponding experimental profile by imp foxs, and assessment of amino acid sidechain conformat ...201121963794
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