| prevalence of pfmdr1, pfcrt, pfdhfr and pfdhps mutations associated with drug resistance, in luanda, angola. | malaria is the infectious disease causing the highest morbidity and mortality in angola and due to widespread chloroquine (cq) resistance, the country has recently changed its first-line treatment recommendations for uncomplicated malaria, from cq to artemisinin combination therapies (act) in adults, and sulphadoxine/pyrimethamine (s/p) in pregnant women. loss of sp sensitivity is, however, progressing rapidly in africa and, in this study, were investigated a number of molecular markers associat ... | 2008 | 19014684 |
| contributions of the two accessory subunits, rnaseh2b and rnaseh2c, to the activity and properties of the human rnase h2 complex. | eukaryotic rnase h2 is a heterotrimeric enzyme. here, we show that the biochemical composition and stoichiometry of the human rnase h2 complex is consistent with the properties previously deduced from genetic studies. the catalytic subunit of eukaryotic rnase h2, rnaseh2a, is well conserved and similar to the monomeric prokaryotic rnase hii. in contrast, the rnaseh2b and rnaseh2c subunits from human and saccharomyces cerevisiae share very little homology, although they both form soluble b/c comp ... | 2009 | 19015152 |
| contributions of the two accessory subunits, rnaseh2b and rnaseh2c, to the activity and properties of the human rnase h2 complex. | eukaryotic rnase h2 is a heterotrimeric enzyme. here, we show that the biochemical composition and stoichiometry of the human rnase h2 complex is consistent with the properties previously deduced from genetic studies. the catalytic subunit of eukaryotic rnase h2, rnaseh2a, is well conserved and similar to the monomeric prokaryotic rnase hii. in contrast, the rnaseh2b and rnaseh2c subunits from human and saccharomyces cerevisiae share very little homology, although they both form soluble b/c comp ... | 2009 | 19015152 |
| structural and biochemical studies of tigar (tp53-induced glycolysis and apoptosis regulator). | activation of the p53 tumor suppressor by cellular stress leads to variable responses ranging from growth inhibition to apoptosis. tigar is a novel p53-inducible gene that inhibits glycolysis by reducing cellular levels of fructose-2,6-bisphosphate, an activator of glycolysis and inhibitor of gluconeogenesis. here we describe structural and biochemical studies of tigar from danio rerio. the overall structure forms a histidine phosphatase fold with a phosphate molecule coordinated to the catalyti ... | 2009 | 19015259 |
| revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein l22. | bacterial antibiotic resistance can occur by many mechanisms. an intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. for example, a 3-residue deletion (deltamkr) in ribosomal protein l22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. here, however, we demonstrate that deltamkr and wild-type ribosomes show comparab ... | 2008 | 19015512 |
| transposition of an insertion sequence, istth7, in the genome of the extreme thermophile thermus thermophilus hb8. | we have identified an active insertion sequence (is) in the genome of thermus thermophilus hb8. transposition was detected as insertional inactivation of a 16s rrna methyltransferase gene, rsmg, resulting in streptomycin resistance. the is element, istth7, is 1029 bp in length, encodes an imperfect 12 bp inverted repeat, and produces a 9 bp direct repeat of the target sequence. the sequence of a putative transposase encoded by istth7 indicates that it is a member of the is427 group within the is ... | 2008 | 19016874 |
| recr-mediated modulation of recf dimer specificity for single- and double-stranded dna. | recf pathway proteins play an important role in the restart of stalled replication and dna repair in prokaryotes. following dna damage, recf, recr, and reco initiate homologous recombination (hr) by loading of the reca recombinase on single-stranded (ss) dna, protected by ssdna-binding protein. the specific role of recf in this process is not well understood. previous studies have proposed that recf directs the recor complex to boundaries of damaged dna regions by recognizing single-stranded/dou ... | 2009 | 19017635 |
| novel escherichia coli rf1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins kid and rele. | novel mutations in prfa, the gene for the polypeptide release factor rf1 of escherichia coli, were isolated using a positive genetic screen based on the pard (kis, kid) toxin-antitoxin system. this original approach allowed the direct selection of mutants with altered translational termination efficiency at uag codons. the isolated prfa mutants displayed a approximately 10-fold decrease in uag termination efficiency with no significant changes in rf1 stability in vivo. all three mutations, g121s ... | 2009 | 19019162 |
| phylogenetic analysis of rubella virus strains from an outbreak in madrid, spain, from 2004 to 2005. | an outbreak of rubella affected 460 individuals in 2004 and 2005 in the community of madrid, spain. most of the patients were nonvaccinated latin american immigrants or spanish males. this study presents the first data on rubella virus genotypes in spain. forty selected clinical samples (2 urine, 5 serum, 3 blood, 2 saliva, and 28 pharyngeal exudate samples) from 40 cases were collected. the 739-nucleotide sequence recommended by the world health organization obtained from viral rna in these sam ... | 2009 | 19020066 |
| phylogenetic analysis of rubella virus strains from an outbreak in madrid, spain, from 2004 to 2005. | an outbreak of rubella affected 460 individuals in 2004 and 2005 in the community of madrid, spain. most of the patients were nonvaccinated latin american immigrants or spanish males. this study presents the first data on rubella virus genotypes in spain. forty selected clinical samples (2 urine, 5 serum, 3 blood, 2 saliva, and 28 pharyngeal exudate samples) from 40 cases were collected. the 739-nucleotide sequence recommended by the world health organization obtained from viral rna in these sam ... | 2009 | 19020066 |
| structure and function of hiv-1 reverse transcriptase: molecular mechanisms of polymerization and inhibition. | the rapid replication of hiv-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. in the absence of an effective vaccine, drugs are the only useful treatment. anti-hiv drugs work; so far drug therapy has saved more than three million years of life. unfortunately, hiv-1 develops resistance to all of the available drugs. although a number of useful anti-hiv drugs have been ... | 2009 | 19022262 |
| structure and function of hiv-1 reverse transcriptase: molecular mechanisms of polymerization and inhibition. | the rapid replication of hiv-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. in the absence of an effective vaccine, drugs are the only useful treatment. anti-hiv drugs work; so far drug therapy has saved more than three million years of life. unfortunately, hiv-1 develops resistance to all of the available drugs. although a number of useful anti-hiv drugs have been ... | 2009 | 19022262 |
| glucose- and glucokinase-controlled mal gene expression in escherichia coli. | malt is the central transcriptional activator of all mal genes in escherichia coli. its activity is controlled by the inducer maltotriose. it can be inhibited by the interaction with certain proteins, and its expression can be controlled. we report here a novel aspect of mal gene regulation: the effect of cytoplasmic glucose and glucokinase (glk) on the activity and the expression of malt. amylomaltase (malq) is essential for the metabolism of maltose. it forms maltodextrins and glucose from mal ... | 2009 | 19028900 |
| glucose- and glucokinase-controlled mal gene expression in escherichia coli. | malt is the central transcriptional activator of all mal genes in escherichia coli. its activity is controlled by the inducer maltotriose. it can be inhibited by the interaction with certain proteins, and its expression can be controlled. we report here a novel aspect of mal gene regulation: the effect of cytoplasmic glucose and glucokinase (glk) on the activity and the expression of malt. amylomaltase (malq) is essential for the metabolism of maltose. it forms maltodextrins and glucose from mal ... | 2009 | 19028900 |
| biochemical and structural properties of mouse kynurenine aminotransferase iii. | kynurenine aminotransferase iii (kat iii) has been considered to be involved in the production of mammalian brain kynurenic acid (kyna), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. the enzyme was identified based on its high sequence identity with mammalian kat i, but its activity toward kynurenine and its structural characteristics have not been established. in this study, the biochemical and structural properties of mouse kat iii (m ... | 2009 | 19029248 |
| biochemical and structural properties of mouse kynurenine aminotransferase iii. | kynurenine aminotransferase iii (kat iii) has been considered to be involved in the production of mammalian brain kynurenic acid (kyna), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. the enzyme was identified based on its high sequence identity with mammalian kat i, but its activity toward kynurenine and its structural characteristics have not been established. in this study, the biochemical and structural properties of mouse kat iii (m ... | 2009 | 19029248 |
| rrna suppressor of a eukaryotic translation initiation factor 5b/initiation factor 2 mutant reveals a binding site for translational gtpases on the small ribosomal subunit. | the translational gtpases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. mutations that impair gtp hydrolysis by eukaryotic translation initiation factor 5b/initiation factor 2 (eif5b/if2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. a mutation in helix h5 of the 18s rrna in the 40s ribosomal subunit and intragenic mutations in domain ii of eif5b suppress the toxic effects associated wit ... | 2009 | 19029250 |
| rrna suppressor of a eukaryotic translation initiation factor 5b/initiation factor 2 mutant reveals a binding site for translational gtpases on the small ribosomal subunit. | the translational gtpases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. mutations that impair gtp hydrolysis by eukaryotic translation initiation factor 5b/initiation factor 2 (eif5b/if2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. a mutation in helix h5 of the 18s rrna in the 40s ribosomal subunit and intragenic mutations in domain ii of eif5b suppress the toxic effects associated wit ... | 2009 | 19029250 |
| function and ribosomal localization of aif6, a translational regulator shared by archaea and eukarya. | the translation factor if6 is shared by the archaea and the eukarya, but is not found in bacteria. the properties of eukaryal if6 (eif6) have been extensively studied, but remain somewhat elusive. eif6 behaves as a ribosome-anti-association factor and is involved in mirna-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. here we have determined the function and ribosomal localization of the archaeal (sulfolobus solfataricus) if6 homologue (aif6). we ... | 2009 | 19036786 |
| function and ribosomal localization of aif6, a translational regulator shared by archaea and eukarya. | the translation factor if6 is shared by the archaea and the eukarya, but is not found in bacteria. the properties of eukaryal if6 (eif6) have been extensively studied, but remain somewhat elusive. eif6 behaves as a ribosome-anti-association factor and is involved in mirna-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. here we have determined the function and ribosomal localization of the archaeal (sulfolobus solfataricus) if6 homologue (aif6). we ... | 2009 | 19036786 |
| minor changes largely restore catalytic activity of archaeal rnase p rna from methanothermobacter thermoautotrophicus. | the increased protein proportion of archaeal and eukaryal ribonuclease (rnase) p holoenzymes parallels a vast decrease in the catalytic activity of their rna subunits (p rnas) alone. we show that a few mutations toward the bacterial p rna consensus substantially activate the catalytic (c-) domain of archaeal p rna from methanothermobacter, in the absence and presence of the bacterial rnase p protein. large increases in ribozyme activity required the cooperative effect of at least two structural ... | 2009 | 19036794 |
| minor changes largely restore catalytic activity of archaeal rnase p rna from methanothermobacter thermoautotrophicus. | the increased protein proportion of archaeal and eukaryal ribonuclease (rnase) p holoenzymes parallels a vast decrease in the catalytic activity of their rna subunits (p rnas) alone. we show that a few mutations toward the bacterial p rna consensus substantially activate the catalytic (c-) domain of archaeal p rna from methanothermobacter, in the absence and presence of the bacterial rnase p protein. large increases in ribozyme activity required the cooperative effect of at least two structural ... | 2009 | 19036794 |
| common thiolation mechanism in the biosynthesis of trna thiouridine and sulphur-containing cofactors. | 2-thioribothymidine (s(2)t), a modified uridine, is found at position 54 in transfer rnas (trnas) from several thermophiles; s(2)t stabilizes the l-shaped structure of trna and is essential for growth at higher temperatures. here, we identified an atpase (trna-two-thiouridine c, ttuc) required for the 2-thiolation of s(2)t in thermus thermophilus and examined in vitro s(2)t formation by ttuc and previously identified s(2)t-biosynthetic proteins (ttua, ttub, and cysteine desulphurases). the c-ter ... | 2008 | 19037260 |
| structure and non-essential function of glycerol kinase in plasmodium falciparum blood stages. | malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). microarray analysis identified glycerol kinase (gk) as the second most highly upregulated gene in plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. phosphorylation of glycerol by gk is the rate-limiting step in glycerol utilization. deletion of this gene from p. falcipa ... | 2009 | 19040641 |
| urm1 at the crossroad of modifications. 'protein modifications: beyond the usual suspects' review series. | the ubiquitin-like protein urm1 can be covalently conjugated to other proteins, such as the yeast thioredoxin peroxidase protein ahp1p, through a mechanism involving the ubiquitin e1-like enzyme uba4. recent findings have revealed a second function of urm1 as a sulphur carrier in the thiolation of eukaryotic cytoplasmic transfer rnas (trnas). interestingly, this new role of urm1 is similar to the sulphur-carrier activity of its prokaryotic counterparts, strengthening the hypothesis that urm1 is ... | 2008 | 19047990 |
| a novel dimerization motif in the c-terminal domain of the thermus thermophilus dead box helicase hera confers substantial flexibility. | dead box helicases are involved in nearly all aspects of rna metabolism. they share a common helicase core, and may comprise additional domains that contribute to rna binding. the thermus thermophilus helicase hera is the first dimeric dead box helicase. crystal structures of hera fragments reveal a bipartite c-terminal domain with a novel dimerization motif and an rna-binding module. we provide a first glimpse on the additional rna-binding module outside the hera helicase core. the dimerization ... | 2009 | 19050012 |
| purification, crystallization and x-ray structures of the two manganese superoxide dismutases from caenorhabditis elegans. | caenorhabditis elegans expresses two manganese superoxide dismutase enzymes (mnsod-2 and mnsod-3) that are targeted to the mitochondrion. mnsod-2 is constitutively expressed, while synthesis of mnsod-3 is inducible. the structures of these two mononuclear metalloenzymes have been determined to 1.8 and 1.7 a resolution, respectively. pink crystals formed in space group p4(1)2(1)2 for each, with unit-cell parameters a = b = 81.0, c = 137.4 a for mnsod-2 and a = b = 81.8, c = 136.0 a for mnsod-3. t ... | 2008 | 19052361 |
| cloning, expression, crystallization and preliminary x-ray crystallographic analysis of 3-dehydroquinate synthase, xoo1243, from xanthomonas oryzae pv. oryzae. | the disease bacterial blight results in serious production losses of rice in asian countries. the arob gene encoding dehydroquinate synthase (dhqs), which is a potential antibiotic target, was identified from the plant-pathogenic bacterium xanthomonas oryzae pv. oryzae (xoo). dhqs plays an essential role in the synthesis of aromatic compounds in the shikimate pathway. the arob gene (xoo1243) was cloned from xoo and the corresponding dhqs protein was subsequently overexpressed in escherichia coli ... | 2008 | 19052366 |
| crystallization and preliminary x-ray diffraction studies of the prototypal homologue of mitoneet (tth-neet0026) from the extreme thermophile thermus thermophilus hb8. | mitoneet (a mammalian mitochondrial outer membrane protein) is a potential pharmacological and clinical target of the insulin-sensitizer pioglitazone. the thermophilic homologue of mitoneet (ttha0026) from thermus thermophilus hb8 has been heterologously overproduced in escherichia coli and purified as a water-soluble prototypal protein containing the mitoneet-like [2fe-2s] cluster. the resultant recombinant protein, named tth-neet0026, has been crystallized in its oxidized form by the hanging-d ... | 2008 | 19052371 |
| the 1.6 a crystal structure of mycobacterium smegmatis mshc: the penultimate enzyme in the mycothiol biosynthetic pathway. | mycobacterium smegmatis mshc catalyzes the atp-dependent condensation of glcn-ins and l-cysteine to form l-cys-glcn-ins, the penultimate step in mycothiol biosynthesis. attempts to crystallize the native, full-length mshc have been unsuccessful. however, incubation of the enzyme with the cysteinyl adenylate analogue, 5'-o-[n-(l-cysteinyl)-sulfamonyl]adenosine (csa), followed by a 24-h limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. the three-dimensional struc ... | 2008 | 19053270 |
| recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from pyrococcus furiosus. | alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-d-glucose-1-phosphate. we report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (pf1535) from a hyperthermophilic archaeon, pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. the apparent 98 kda recombinant enzyme was active over a broad range of temperatures and ... | 2008 | 19054743 |
| recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from pyrococcus furiosus. | alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-d-glucose-1-phosphate. we report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (pf1535) from a hyperthermophilic archaeon, pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. the apparent 98 kda recombinant enzyme was active over a broad range of temperatures and ... | 2008 | 19054743 |
| identification of a new endogenous metabolite and the characterization of its protein interactions through an immobilization approach. | the emerging field of global mass-based metabolomics provides a platform for discovering unknown metabolites and their specific biochemical pathways. we report the identification of a new endogenous metabolite, n(4)-(n-acetylaminopropyl)spermidine and the use of a novel proteomics based method for the investigation of its protein interaction using metabolite immobilization on agarose beads. the metabolite was isolated from the organism pyrococcus furiosus, and structurally characterized through ... | 2009 | 19055353 |
| bridge helix and trigger loop perturbations generate superactive rna polymerases. | cellular rna polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. two domains in particular, the bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. the functional relevance of these structural changes has been difficult to assess from the relatively small number of static crystal ... | 2008 | 19055851 |
| differential effects of mitochondrial complex i inhibitors on production of reactive oxygen species. | we have investigated the production of reactive oxygen species (ros) by complex i in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of nadh, by means of the probe 2',7'-dichlorodihydrofluorescein diacetate. ros production by complex i is strictly related to its inhibited state. our results indicate that different complex i inhibitors can be grouped into two classes: class a inhibitors (rotenone, piericidin a and rolliniastatin 1 and 2) ... | 2009 | 19059197 |
| differential effects of mitochondrial complex i inhibitors on production of reactive oxygen species. | we have investigated the production of reactive oxygen species (ros) by complex i in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of nadh, by means of the probe 2',7'-dichlorodihydrofluorescein diacetate. ros production by complex i is strictly related to its inhibited state. our results indicate that different complex i inhibitors can be grouped into two classes: class a inhibitors (rotenone, piericidin a and rolliniastatin 1 and 2) ... | 2009 | 19059197 |
| superoxide dismutase from the eukaryotic thermophile alvinella pompejana: structures, stability, mechanism, and insights into amyotrophic lateral sclerosis. | prokaryotic thermophiles supply stable human protein homologs for structural biology; yet, eukaryotic thermophiles would provide more similar macromolecules plus those missing in microbes. alvinella pompejana is a deep-sea hydrothermal-vent worm that has been found in temperatures averaging as high as 68 degrees c, with spikes up to 84 degrees c. here, we used cu,zn superoxide dismutase (sod) to test if this eukaryotic thermophile can provide insights into macromolecular mechanisms and stability ... | 2009 | 19063897 |
| superoxide dismutase from the eukaryotic thermophile alvinella pompejana: structures, stability, mechanism, and insights into amyotrophic lateral sclerosis. | prokaryotic thermophiles supply stable human protein homologs for structural biology; yet, eukaryotic thermophiles would provide more similar macromolecules plus those missing in microbes. alvinella pompejana is a deep-sea hydrothermal-vent worm that has been found in temperatures averaging as high as 68 degrees c, with spikes up to 84 degrees c. here, we used cu,zn superoxide dismutase (sod) to test if this eukaryotic thermophile can provide insights into macromolecular mechanisms and stability ... | 2009 | 19063897 |
| crystal structure of a translation termination complex formed with release factor rf2. | we report the crystal structure of a translation termination complex formed by the thermus thermophilus 70s ribosome bound with release factor rf2, in response to a uaa stop codon, solved at 3 a resolution. the backbone of helix alpha5 and the side chain of serine of the conserved spf motif of rf2 recognize u1 and a2 of the stop codon, respectively. a3 is unstacked from the first 2 bases, contacting thr-216 and val-203 of rf2 and stacking on g530 of 16s rrna. the structure of the rf2 complex sup ... | 2008 | 19064930 |
| vectorial proton transfer coupled to reduction of o2 and no by a heme-copper oxidase. | the heme-copper oxidase (hcuo) superfamily consists of integral membrane proteins that catalyze the reduction of either oxygen or nitric oxide. the hcuos that reduce o(2) to h(2)o couple this reaction to the generation of a transmembrane proton gradient by using electrons and protons from opposite sides of the membrane and by pumping protons from inside the cell or organelle to the outside. the bacterial no-reductases (nor) reduce no to n(2)o (2no + 2e(-) + 2h(+) --> n(2)o + h(2)o), a reaction a ... | 2008 | 19074284 |
| structure and functional role of dynein's microtubule-binding domain. | dynein motors move various cargos along microtubules within the cytoplasm and power the beating of cilia and flagella. an unusual feature of dynein is that its microtubule-binding domain (mtbd) is separated from its ring-shaped aaa+ adenosine triphosphatase (atpase) domain by a 15-nanometer coiled-coil stalk. we report the crystal structure of the mouse cytoplasmic dynein mtbd and a portion of the coiled coil, which supports a mechanism by which the atpase domain and mtbd may communicate through ... | 2008 | 19074350 |
| heterogeneity of large macromolecular complexes revealed by 3d cryo-em variance analysis. | macromolecular structure determination by cryo-electron microscopy (em) and single-particle analysis are based on the assumption that imaged molecules have identical structure. with the increased size of processed data sets, it becomes apparent that many complexes coexist in a mixture of conformational states or contain flexible regions. we describe an implementation of the bootstrap resampling technique that yields estimates of voxel-by-voxel variance of a structure reconstructed from the set o ... | 2008 | 19081053 |
| structural basis for inactivation of the human pyruvate dehydrogenase complex by phosphorylation: role of disordered phosphorylation loops. | we report the crystal structures of the phosporylated pyruvate dehydrogenase (e1p) component of the human pyruvate dehydrogenase complex (pdc). the complete phosphorylation at ser264-alpha (site 1) of a variant e1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the pdc core. we show that unlike its unmodified counterpart, the presence of a phosphoryl group at ser264-alpha prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three ph ... | 2008 | 19081061 |
| effect of simulated microgravity on oxidation-sensitive gene expression in pc12 cells. | oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). a baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. the objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (pc12)] under simulated micro-g conditions. the pc12 cell line is well cha ... | 2006 | 19081771 |
| structure of an argonaute silencing complex with a seed-containing guide dna and target rna duplex. | here we report on a 3.0 a crystal structure of a ternary complex of wild-type thermus thermophilus argonaute bound to a 5'-phosphorylated 21-nucleotide guide dna and a 20-nucleotide target rna containing cleavage-preventing mismatches at the 10-11 step. the seed segment (positions 2 to 8) adopts an a-helical-like watson-crick paired duplex, with both ends of the guide strand anchored in the complex. an arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in a ... | 2008 | 19092929 |
| a signal relay between ribosomal protein s12 and elongation factor ef-tu during decoding of mrna. | codon recognition by aminoacyl-trna on the ribosome triggers a process leading to gtp hydrolysis by elongation factor tu (ef-tu) and release of aminoacyl-trna into the a site of the ribosome. the nature of this signal is largely unknown. here, we present genetic evidence that a specific set of direct interactions between ribosomal protein s12 and aminoacyl-trna, together with contacts between s12 and 16s rrna, provide a pathway for the signaling of codon recognition to ef-tu. three novel amino a ... | 2009 | 19095621 |
| mitochondrial dna damage in iron overload. | chronic iron overload has slow and insidious effects on heart, liver, and other organs. because iron-driven oxidation of most biologic materials (such as lipids and proteins) is readily repaired, this slow progression of organ damage implies some kind of biological "memory." we hypothesized that cumulative iron-catalyzed oxidant damage to mtdna might occur in iron overload, perhaps explaining the often lethal cardiac dysfunction. real time pcr was used to examine the "intactness" of mttdna in cu ... | 2009 | 19095657 |
| functional role of coenzyme q in the energy coupling of nadh-coq oxidoreductase (complex i): stabilization of the semiquinone state with the application of inside-positive membrane potential to proteoliposomes. | coenzyme q10 (which is also designated as coq10, ubiquinone-10, uq10, coq, uq or simply as q) plays an important role in energy metabolism. for nadh-q oxidoreductase (complex i), ohnishi and salerno proposed a hypothesis that the proton pump is operated by the redox-driven conformational change of a q-binding protein, and that the bound form of semiquinone (sq) serves as its gate [febs letters 579 (2005) 45-55]. this was based on the following experimental results: (i) epr signals of the fast-re ... | 2008 | 19096096 |
| functional specialization of transcription elongation factors. | elongation factors nusg and rfah evolved from a common ancestor and utilize the same binding site on rna polymerase (rnap) to modulate transcription. however, although nusg associates with rnap transcribing most escherichia coli genes, rfah regulates just a few operons containing ops, a dna sequence that mediates rfah recruitment. here, we describe the mechanism by which this specificity is maintained. we observe that rfah action is indeed restricted to those several operons that are devoid of n ... | 2009 | 19096362 |
| functional specialization of transcription elongation factors. | elongation factors nusg and rfah evolved from a common ancestor and utilize the same binding site on rna polymerase (rnap) to modulate transcription. however, although nusg associates with rnap transcribing most escherichia coli genes, rfah regulates just a few operons containing ops, a dna sequence that mediates rfah recruitment. here, we describe the mechanism by which this specificity is maintained. we observe that rfah action is indeed restricted to those several operons that are devoid of n ... | 2009 | 19096362 |
| a novel insertion mutation in streptomyces coelicolor ribosomal s12 protein results in paromomycin resistance and antibiotic overproduction. | we identified a novel paromomycin resistance-associated mutation in rpsl, caused by the insertion of a glycine residue at position 92, in streptomyces coelicolor ribosomal protein s12. this insertion mutation (gi92) resulted in a 20-fold increase in the paromomycin resistance level. in combination with another s12 mutation, k88e, the gi92 mutation markedly enhanced the production of the blue-colored polyketide antibiotic actinorhodin and the red-colored antibiotic undecylprodigiosin. the gene re ... | 2009 | 19104019 |
| a novel insertion mutation in streptomyces coelicolor ribosomal s12 protein results in paromomycin resistance and antibiotic overproduction. | we identified a novel paromomycin resistance-associated mutation in rpsl, caused by the insertion of a glycine residue at position 92, in streptomyces coelicolor ribosomal protein s12. this insertion mutation (gi92) resulted in a 20-fold increase in the paromomycin resistance level. in combination with another s12 mutation, k88e, the gi92 mutation markedly enhanced the production of the blue-colored polyketide antibiotic actinorhodin and the red-colored antibiotic undecylprodigiosin. the gene re ... | 2009 | 19104019 |
| epr evidence of cyanide binding to the mn(mg) center of cytochrome c oxidase: support for cu(a)-mg involvement in proton pumping. | we examined the anion binding behavior of the mg(mn) site in cytochrome c oxidase to test a possible role of this center in proton pumping. rhodobacter sphaeroides grown in a mn(ii)-rich medium replaces the intrinsic mg(ii) ion with an epr-detectable mn(ii) ion without change in activity. due to its close proximity and a shared ligand, oxidized cu(a) is spin-coupled to the mn(ii) ion, affecting the epr spectrum. an examination of both bovine and r.s. oxidase crystal structures reveals a hydrogen ... | 2009 | 19108635 |
| characterization of two novel alpha-glucosidases from bifidobacterium breve ucc2003. | two alpha-glucosidase-encoding genes (agl1 and agl2) from bifidobacterium breve ucc2003 were identified and characterized. based on their similarity to characterized carbohydrate hydrolases, the agl1 and agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the alpha-1,6-glucosidases (ec 3.2.1.10). recombinant agl1 and agl2 into which a his(12) sequence was incorporated (agl1(his) and agl2(his), respectively) exhibited hydrolytic activity towards panose, isomaltose, i ... | 2009 | 19114534 |
| characterization of two novel alpha-glucosidases from bifidobacterium breve ucc2003. | two alpha-glucosidase-encoding genes (agl1 and agl2) from bifidobacterium breve ucc2003 were identified and characterized. based on their similarity to characterized carbohydrate hydrolases, the agl1 and agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the alpha-1,6-glucosidases (ec 3.2.1.10). recombinant agl1 and agl2 into which a his(12) sequence was incorporated (agl1(his) and agl2(his), respectively) exhibited hydrolytic activity towards panose, isomaltose, i ... | 2009 | 19114534 |
| the crystal structure of galacto-n-biose/lacto-n-biose i phosphorylase: a large deformation of a tim barrel scaffold. | galacto-n-biose/lacto-n-biose i phosphorylase (glnbp) from bifidobacterium longum, a key enzyme for intestinal growth, phosphorolyses galacto-n-biose and lacto-n-biose i with anomeric inversion. glnbp homologues are often found in human pathogenic and commensal bacteria, and their substrate specificities potentially define the nutritional acquisition ability of these microbes in their habitat. we report the crystal structures of glnbp in five different ligand-binding forms. this is the first thr ... | 2009 | 19124470 |
| architectural underpinnings of the genetic code for glutamine. | structure-based mutational analysis was used to probe the architecture of the glutamine binding pocket in escherichia coli glutaminyl-trna synthetase (glnrs). crystallographic studies of several different glnrs complexes in a lattice that supports catalytic activity have shown that the glutamine amide group makes only ambiguous hydrogen-bonding interactions with a tyrosine hydroxyl and bound water molecule, rather than the highly specific hydrogen-bonding and electrostatic interactions made by t ... | 2009 | 19128026 |
| dihydroorotase from the hyperthermophile aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis. | in prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (cps), aspartate transcarbamoylase (atc), and dihydroorotase (dho), are commonly expressed separately and either function independently (escherichia coli) or associate into multifunctional complexes (aquifex aeolicus). in mammals the enzymes are expressed as a single polypeptide chain (cad) in the order cps-dho-atc and associate into a hexamer. this study presents the three-dimensional structure of ... | 2009 | 19128030 |
| ribosome hijacking: a role for small protein b during trans-translation. | tight recognition of codon-anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. in eubacteria, translational surveillance and ribosome rescue are performed by the 'tmrna-smpb' system (transfer messenger rna-small protein b). remarkably, entry and accommodation of aminoacylated-tmrna into stalled ribosomes occur without a codon-anticodon interaction but in the presence of smpb. here, we show that within a stalled ribosome, smpb interacts with the three univer ... | 2009 | 19132006 |
| combined microspectrophotometric and crystallographic examination of chemically reduced and x-ray radiation-reduced forms of cytochrome ba3 oxidase from thermus thermophilus: structure of the reduced form of the enzyme. | three paths for obtaining crystals of reduced (ii-e4q/i-k258r) cytochrome ba(3) are described, and the structures of these are reported at approximately 2.8-3.0 a resolution. microspectrophotometry of single crystals of thermus ba(3) oxidase at 100 k was used to show that crystals of the oxidized enzyme are reduced in an intense x-ray (beam line 7-1 at the stanford synchrotron radiation laboratory), being nearly complete in 1 min. the previously reported structures of ba(3) (protein data bank en ... | 2009 | 19140675 |
| a conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate. | proline dehydrogenase (prodh) catalyzes the oxidation of l-proline to delta-1-pyrroline-5-carboxylate. prodhs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-l-proline) as the substrate, but the basis for specificity is unknown. the goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how prodhs discriminate between the two closely related molecules, prol ... | 2009 | 19140736 |
| coarse-grained modeling of large rna molecules with knowledge-based potentials and structural filters. | understanding the function of complex rna molecules depends critically on understanding their structure. however, creating three-dimensional (3d) structural models of rna remains a significant challenge. we present a protocol (the nucleic acid simulation tool [nast]) for rna modeling that uses an rna-specific knowledge-based potential in a coarse-grained molecular dynamics engine to generate plausible 3d structures. we demonstrate nast's capabilities by using only secondary structure and tertiar ... | 2009 | 19144906 |
| genetic and structural analysis of base substitutions in the central pseudoknot of thermus thermophilus 16s ribosomal rna. | characterization of base substitutions in rrnas has provided important insights into the mechanism of protein synthesis. knowledge of the structural effects of such alterations is limited, and could be greatly expanded with the development of a genetic system based on an organism amenable to both genetics and structural biology. here, we describe the genetic analysis of base substitutions in 16s ribosomal rna of the extreme thermophile thermus thermophilus, and an analysis of the conformational ... | 2009 | 19144908 |
| electronic structure of the ground and excited states of the cu(a) site by nmr spectroscopy. | the electronic properties of thermus thermophilus cu(a) in the oxidized form were studied by (1)h and (13)c nmr spectroscopy. all of the (1)h and (13)c resonances from cysteine and imidazole ligands were observed and assigned in a sequence-specific fashion. the detection of net electron spin density on a peptide moiety is attributed to the presence of a h-bond to a coordinating sulfur atom. this hydrogen bond is conserved in all natural cu(a) variants and plays an important role for maintaining ... | 2009 | 19146411 |
| proteomic analysis of protein tyrosine nitration after ischemia reperfusion injury: mitochondria as the major target. | endothelial nitric oxide synthase-derived no and its derivative, peroxynitrite (onoo(-)), suppresses oxygen consumption by nitration of mitochondrial proteins after reperfusion. however, very few nitrated proteins are identified to date. in this paper, ischemia/reperfusion (i/r) injury was induced in mouse heart by ligation and release of the left anterior descending coronary artery. western blotting showed that tyrosine nitration was higher in i/r hearts. nitrated proteins were identified by ca ... | 2009 | 19150419 |
| proteomic analysis of protein tyrosine nitration after ischemia reperfusion injury: mitochondria as the major target. | endothelial nitric oxide synthase-derived no and its derivative, peroxynitrite (onoo(-)), suppresses oxygen consumption by nitration of mitochondrial proteins after reperfusion. however, very few nitrated proteins are identified to date. in this paper, ischemia/reperfusion (i/r) injury was induced in mouse heart by ligation and release of the left anterior descending coronary artery. western blotting showed that tyrosine nitration was higher in i/r hearts. nitrated proteins were identified by ca ... | 2009 | 19150419 |
| mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at trna wobble positions. | the wobble modification in trnas, 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)u), is required for the proper decoding of nnr codons in eukaryotes. the 2-thio group confers conformational rigidity of mcm(5)s(2)u by largely fixing the c3'-endo ribose puckering, ensuring stable and accurate codon-anticodon pairing. we have identified five genes in saccharomyces cerevisiae, yil008w (urm1), yhr111w (uba4), yor251c (tum1), ynl119w (ncs2) and ygl211w (ncs6), that are required for 2-thiolation of m ... | 2009 | 19151091 |
| cloning, expression, crystallization and preliminary x-ray crystallographic analysis of glutamyl-trna synthetase (xoo1504) from xanthomonas oryzae pv. oryzae. | the gltx gene from xanthomonas oryzae pv. oryzae (xoo1504) encodes glutamyl-trna synthetase (glurs), one of the most important enzymes involved in bacterial blight (bb), which causes huge production losses of rice worldwide. glurs is a class i-type aminoacyl-trna synthetase (aars) that is primarily responsible for the glutamylation of trna(glu). it plays an essential role in protein synthesis, as well as the regulation of cells, in all organisms. as it represents an important target for the deve ... | 2009 | 19153456 |
| cloning, expression, crystallization and preliminary x-ray crystallographic analysis of glutamyl-trna synthetase (xoo1504) from xanthomonas oryzae pv. oryzae. | the gltx gene from xanthomonas oryzae pv. oryzae (xoo1504) encodes glutamyl-trna synthetase (glurs), one of the most important enzymes involved in bacterial blight (bb), which causes huge production losses of rice worldwide. glurs is a class i-type aminoacyl-trna synthetase (aars) that is primarily responsible for the glutamylation of trna(glu). it plays an essential role in protein synthesis, as well as the regulation of cells, in all organisms. as it represents an important target for the deve ... | 2009 | 19153456 |
| x-ray diffraction analysis of a human trna(gly) acceptor-stem microhelix isoacceptor at 1.18 a resolution. | interest has been focused on comparative x-ray structure analyses of different trna(gly) acceptor-stem helices. trna(gly)/glycyl-trna synthetase belongs to the so-called class ii system, in which the trna identity elements consist of simple and unique determinants that are located in the trna acceptor stem and the discriminator base. comparative structure investigations of trna(gly) microhelices provide insight into the role of trna identity elements. predominant differences in the structures of ... | 2009 | 19153458 |
| x-ray diffraction analysis of a human trna(gly) acceptor-stem microhelix isoacceptor at 1.18 a resolution. | interest has been focused on comparative x-ray structure analyses of different trna(gly) acceptor-stem helices. trna(gly)/glycyl-trna synthetase belongs to the so-called class ii system, in which the trna identity elements consist of simple and unique determinants that are located in the trna acceptor stem and the discriminator base. comparative structure investigations of trna(gly) microhelices provide insight into the role of trna identity elements. predominant differences in the structures of ... | 2009 | 19153458 |
| characterization of a heat-stable enzyme possessing gtp-dependent rna ligase activity from a hyperthermophilic archaeon, pyrococcus furiosus. | using an expression protein library of a hyperthermophilic archaeon, pyrococcus furiosus, we identified a gene (pf0027) that encodes a protein with heat-stable cyclic nucleotide phosphodiesterase (cpdase) activity. the pf0027 gene encoded a 21-kda protein and an amino acid sequence that showed approximately 27% identity to that of the 2'-5' trna ligase protein, ligt (20 kda), from escherichia coli. we found that the purified pf0027 protein possessed gtp-dependent rna ligase activity and that syn ... | 2009 | 19155324 |
| new role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase. | using fmn and a reducing agent such as nad(p)h, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphat ... | 2009 | 19158086 |
| a novel bicistronic vector for overexpressing mycobacterium tuberculosis proteins in escherichia coli. | a putative dna glycosylase encoded by the rv3297 gene (mtunei2) has been identified in mycobacterium tuberculosis. our efforts to express this gene in escherichia coli either by supplementing trnas for rare codons or optimizing the gene with preferred codons for e. coli resulted in little or no expression. on the other hand, high-level expression was observed using a bicistronic expression vector in which the target gene was translationally coupled to an upstream leader sequence. further compari ... | 2009 | 19162193 |
| a novel bicistronic vector for overexpressing mycobacterium tuberculosis proteins in escherichia coli. | a putative dna glycosylase encoded by the rv3297 gene (mtunei2) has been identified in mycobacterium tuberculosis. our efforts to express this gene in escherichia coli either by supplementing trnas for rare codons or optimizing the gene with preferred codons for e. coli resulted in little or no expression. on the other hand, high-level expression was observed using a bicistronic expression vector in which the target gene was translationally coupled to an upstream leader sequence. further compari ... | 2009 | 19162193 |
| a peroxide bridge between fe and cu ions in the o2 reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump. | the fully oxidized form of cytochrome c oxidase, immediately after complete oxidation of the fully reduced form, pumps protons upon each of the initial 2 single-electron reduction steps, whereas protons are not pumped during single-electron reduction of the fully oxidized "as-isolated" form (the fully oxidized form without any reduction/oxidation treatment) [bloch d, et al. (2004) the catalytic cycle of cytochrome c oxidase is not the sum of its two halves. proc natl acad sci usa 101:529-533]. f ... | 2009 | 19164527 |
| a systematic evaluation of the function of the protein-remodeling factor hsp104 in [psi+] prion propagation in s. cerevisiae by comprehensive chromosomal mutations. | the yeast prion [psi(+)] represents an aggregated state of the translational release factor sup35 (erf3) and deprives termination complexes of functional sup35, resulting in nonsense codon suppression. protein-remodeling factor hsp104 is involved in thermotolerance and [psi(+)] propagation, however the structure-and-function relationship of hsp104 for [psi(+)] remains unclear. in this study, we engineered 58 chromosomal hsp104 mutants that affect residues considered structurally or functionally ... | 2007 | 19164920 |
| biosynthesis of undecaprenyl phosphate-galactosamine and undecaprenyl phosphate-glucose in francisella novicida. | lipid a of francisella tularensis subsp. novicida contains a galactosamine (galn) residue linked to its 1-phosphate group. as shown in the preceding paper, this galn unit is transferred to lipid a from the precursor undecaprenyl phosphate-beta-d-galn. a small portion of the free lipid a of francisella novicida is further modified with a glucose residue at position-6'. we now demonstrate that the two f. novicida homologues of escherichia coli arnc, designated flmf1 and flmf2, are essential for li ... | 2009 | 19166326 |
| characterization of oxidized guanosine 5'-triphosphate as a viable inhibitor of soluble guanylyl cyclase. | the guanine base is prone to oxidation by free radicals regardless of the cellular moiety it is bound to. however, under conditions of oxidative stress, 8-oxoguanosine triphosphate (oxo(8)gtp) formation has been shown to occur without oxidation of the guanine base in dna. in vitro studies have suggested that oxo(8)gtp could impact g-protein signaling and rna synthesis. whether increased levels of oxo(8)gtp translate into cellular malfunction is unknown. data presented herein show that oxo(8)gtp ... | 2009 | 19167482 |
| allosteric control of escherichia coli rrna promoter complexes by dksa. | the escherichia coli dksa protein inserts into the rna polymerase (rnap) secondary channel, modifying the transcription initiation complex so that promoters with specific kinetic characteristics are regulated by changes in the concentrations of ppgpp and ntps. we used footprinting assays to determine the specific kinetic intermediate, rp(i), on which dksa acts. genetic approaches identified substitutions in the rnap switch regions, bridge helix, and trigger loop that mimicked, reduced, or enhanc ... | 2009 | 19171784 |
| ribosomal translocation: one step closer to the molecular mechanism. | protein synthesis occurs in ribosomes, the targets of numerous antibiotics. how these large and complex machines read and move along mrna have proven to be challenging questions. in this review, we focus on translocation, the last step of the elongation cycle in which movement of trna and mrna is catalyzed by elongation factor g. translocation entails large-scale movements of the trnas and conformational changes in the ribosome that require numerous tertiary contacts to be disrupted and reformed ... | 2009 | 19173642 |
| molecular modeling and site-directed mutagenesis reveal essential residues for catalysis in a prokaryote-type aspartate aminotransferase. | we recently reported that aspartate (asp) biosynthesis in plant chloroplasts is catalyzed by two different asp aminotransferases (aat): a previously characterized eukaryote type and a prokaryote type (pt-aat) similar to bacterial and archaebacterial enzymes. the available molecular and kinetic data suggest that the eukaryote-type aat is involved in the shuttling of reducing equivalents through the plastidic membrane, whereas the pt-aat could be involved in the biosynthesis of the asp-derived ami ... | 2009 | 19176717 |
| a green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms. | green fluorescent protein (gfp) fusion proteins provide a potentially facile tool for identification of well expressed, properly behaved membrane proteins for biochemical and structural study. here, we present a gfp-expression survey of >300 membrane proteins from 18 bacterial and archaeal extremophiles, organisms expected to be rich sources of membrane proteins having robust biophysical properties. we find that gfp-fusion fluorescence intensity is an excellent indicator of over-expression poten ... | 2009 | 19177357 |
| a green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms. | green fluorescent protein (gfp) fusion proteins provide a potentially facile tool for identification of well expressed, properly behaved membrane proteins for biochemical and structural study. here, we present a gfp-expression survey of >300 membrane proteins from 18 bacterial and archaeal extremophiles, organisms expected to be rich sources of membrane proteins having robust biophysical properties. we find that gfp-fusion fluorescence intensity is an excellent indicator of over-expression poten ... | 2009 | 19177357 |
| constant c10 ring stoichiometry in the escherichia coli atp synthase analyzed by cross-linking. | the subunit c stoichiometry of escherichia coli atp synthase was studied by intermolecular cross-linking via oxidation of bi-cysteine-substituted subunit c (ca21c/cm65c). independent of the carbon source used for growth and independent of the presence of other fof1 subunits, an equal pattern of cross-link formation stopping at the formation of decamers was obtained. | 2009 | 19181809 |
| mechanistic and functional insights into fatty acid activation in mycobacterium tuberculosis. | the recent discovery of fatty acyl-amp ligases (faals) in mycobacterium tuberculosis (mtb) provided a new perspective of fatty acid activation. these proteins convert fatty acids to the corresponding adenylates, which are intermediates of acyl-coa-synthesizing fatty acyl-coa ligases (facls). presently, it is not evident how obligate pathogens such as mtb have evolved such new themes of functional versatility and whether the activation of fatty acids to acyladenylates could indeed be a general me ... | 2009 | 19182784 |
| x-ray crystal structure of garr-tartronate semialdehyde reductase from salmonella typhimurium. | tartronate semialdehyde reductases (tsrs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using nad as cofactor in the final stage of d-glycerate biosynthesis. these enzymes belong to family of structurally and mechanically related beta-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. here, we present the crystal structure of garr a tsr from salmonella typhimurium determi ... | 2009 | 19184529 |
| mechanism of inhibition of the v-type molecular motor by tributyltin chloride. | tributyltin chloride (tbt-cl) is an endocrine disruptor found in many animal species, and it is also known to be an inhibitor for the v-atpases that are emerging as potential targets in the treatment of diseases such as osteoporosis and cancer. we demonstrated by using biochemical and single-molecular imaging techniques that tbt-cl arrests an elementary step for rotary catalysis of the v(1) motor domain. in the presence of tbt-cl, the consecutive rotation of v(1) paused for a long duration ( app ... | 2009 | 19186155 |
| accommodation of two diatomic molecules in cytochrome bo: insights into no reductase activity in terminal oxidases. | bacterial heme-copper terminal oxidases react quickly with no to form a heme-nitrosyl complex, which, in some of these enzymes, can further react with a second no molecule to produce n(2)o. previously, we characterized the heme a(3)-no complex formed in cytochrome ba(3) from thermus thermophilus and the product of its low-temperature illumination. we showed that the photolyzed no group binds to cu(b)(i) to form an end-on no-cu(b) or a side-on copper-nitrosyl complex, which is likely to represent ... | 2009 | 19187032 |
| enhancement of the seed-target recognition step in rna silencing by a piwi/mid domain protein. | target recognition in rna silencing is governed by the "seed sequence" of a guide rna strand associated with the piwi/mid domain of an argonaute protein in risc. using a reconstituted in vitro target recognition system, we show that a model piwi/mid domain protein confers position-dependent tightening and loosening of guide-strand-target interactions. over the seed sequence, the interaction affinity is enhanced up to approximately 300-fold. enhancement is achieved through a reduced entropy penal ... | 2009 | 19187762 |
| an unexpected type of ribosomes induced by kasugamycin: a look into ancestral times of protein synthesis? | translation of leaderless mrnas, lacking ribosomal recruitment signals other than the 5'-terminal aug-initiating codon, occurs in all three domains of life. contemporary leaderless mrnas may therefore be viewed as molecular fossils resembling ancestral mrnas. here, we analyzed the phenomenon of sustained translation of a leaderless mrna in the presence of the antibiotic kasugamycin. unexpected from the known in vitro effects of the drug, kasugamycin induced the formation of stable approximately ... | 2009 | 19187763 |
| evolutionary basis for the coupled-domain motions in thermus thermophilus leucyl-trna synthetase. | aminoacyl-trna synthetases are multidomain proteins that catalyze the covalent attachment of amino acids to their cognate transfer rna. various domains of an aminoacyl-trna synthetase perform their specific functions in a highly coordinated manner to maintain high accuracy in protein synthesis in cells. the coordination of their function, therefore, requires communication between domains. in this study we explored the relevance of enzyme motion in domain-domain communications. specifically, we a ... | 2009 | 19188368 |
| critical roles of subunit nuoh (nd1) in the assembly of peripheral subunits with the membrane domain of escherichia coli ndh-1. | the bacterial proton-translocating nadh:quinone oxidoreductase (ndh-1) consists of two domains, a peripheral arm and a membrane arm. nuoh is a counterpart of nd1, which is one of seven mitochondrially encoded hydrophobic subunits, and is considered to be involved in quinone/inhibitor binding. sequence comparison in a wide range of species showed that nuoh is comprehensively conserved, particularly with charged residues in the cytoplasmic side loops. we have constructed 40 mutants of 27 conserved ... | 2009 | 19189973 |
| a minimized rrna-binding site for ribosomal protein s4 and its implications for 30s assembly. | primary ribosomal protein s4 is essential for 30s ribosome biogenesis in eubacteria, because it nucleates subunit assembly and helps coordinate assembly with the synthesis of its rrna and protein components. s4 binds a five-helix junction (5wj) that bridges the 5' and 3' ends of the 16s 5' domain. to delineate which nucleotides contribute to s4 recognition, sequential deletions of the 16s 5' domain were tested in competitive s4-binding assays based on electrophoretic mobility shifts. s4 binds th ... | 2009 | 19190093 |
| delineation of alternative conformational states in escherichia coli peptide deformylase via thermodynamic studies for the binding of actinonin. | we investigated the binding of a naturally occurring antibiotic, actinonin, to the ni(2+)-reconstituted recombinant form of escherichia coli peptide deformylase (pdf(ec)) via isothermal titration microcalorimetry. the binding data conformed to both exothermic and endothermic phases with magnitudes of deltag degrees , deltah degrees , and tdeltas degrees being equal to -12, -2.7, and 9.3 kcal/mol and -8.7, 3.9, and 12.6 kcal/mol, respectively. evidently, although both phases are dominated by favo ... | 2009 | 19191548 |
| hgf and bmp-7 ameliorate high glucose-induced epithelial-to-mesenchymal transition of peritoneal mesothelium. | over time, peritoneal dialysis results in functional and structural alterations of the peritoneal membrane, but the underlying mechanisms and whether these changes are reversible are not completely understood. here, we studied the effects of high levels of glucose, which are found in the dialysate, on human peritoneal mesothelial cells (hpmcs). we found that high concentrations of glucose induced epithelial-to-mesenchymal transition (emt) of hpmc, suggested by decreased expression of e-cadherin ... | 2009 | 19193779 |
| crystallization and x-ray analysis of human cytoplasmic phenylalanyl-trna synthetase. | human cytosolic phenylalanyl-trna synthetase (hcphers) is responsible for the covalent attachment of phenylalanine to its cognate trna(phe). significant differences between the amino-acid sequences of eukaryotic and prokaryotic pherss indicate that the domain composition of hcphers differs from that of the thermus thermophilus analogue. as a consequence of the absence of the anticodon-recognizing b8 domain, the binding mode of trna(phe) to hcphers is expected to differ from that in prokaryotes. ... | 2009 | 19193993 |
| escherichia coli trna(arg) acceptor-stem isoacceptors: comparative crystallization and preliminary x-ray diffraction analysis. | the aminoacylation of trna is a crucial step in cellular protein biosynthesis. recognition of the cognate trna by the correct aminoacyl-trna synthetase is ensured by trna identity elements. in trna(arg), the identity elements consist of the anticodon, parts of the d-loop and the discriminator base. the minor groove of the aminoacyl stem interacts with the arginyl-trna synthetase. as a consequence of the redundancy of the genetic code, six trna(arg) isoacceptors exist. in the present work, three ... | 2009 | 19193994 |
| crystallization, data collection and data processing of maltose-binding protein (male) from the phytopathogen xanthomonas axonopodis pv. citri. | maltose-binding protein is the periplasmic component of the abc transporter responsible for the uptake of maltose/maltodextrins. the xanthomonas axonopodis pv. citri maltose-binding protein male has been crystallized at 293 k using the hanging-drop vapour-diffusion method. the crystal belonged to the primitive hexagonal space group p6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 a, and contained two molecules in the asymetric unit. it diffracted to 2.24 a resolution. | 2009 | 19193996 |
| preliminary x-ray crystallographic analysis of ornithine acetyltransferase (rv1653) from mycobacterium tuberculosis. | the gene product of open reading frame rv1653 from mycobacterium tuberculosis is annotated as encoding a probable ornithine acetyltransferase (oatase; ec 2.3.1.35), an enzyme that catalyzes two steps in the arginine-biosynthesis pathway. it transfers an acetyl group from n-acetylornithine to l-glutamate to produce n-acetylglutamate and l-ornithine. rv1653 was crystallized using the sitting-drop vapour-diffusion method. the native crystals diffracted to a resolution of 1.7 a and belonged to space ... | 2009 | 19194014 |