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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 ...200919144908
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 ...200919146411
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 ...200919150419
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 ...200919150419
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 ...200919151091
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 ...200919153456
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 ...200919153456
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 ...200919153458
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 ...200919153458
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 ...200919155324
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 ...200919158086
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 ...200919162193
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 ...200919162193
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 ...200919164527
the mycobacterium tuberculosis mep (2c-methyl-d-erythritol 4-phosphate) pathway as a new drug target.tuberculosis (tb) is still a major public health problem, compounded by the human immunodeficiency virus (hiv)-tb co-infection and recent emergence of multidrug-resistant (mdr) and extensively drug resistant (xdr)-tb. novel anti-tb drugs are urgently required. in this context, the 2c-methyl-d-erythritol 4-phosphate (mep) pathway of mycobacterium tuberculosis has drawn attention; it is one of several pathways vital for m. tuberculosis viability and the human host lacks homologous enzymes. thus, t ...200918793870
the mycobacterium tuberculosis mep (2c-methyl-d-erythritol 4-phosphate) pathway as a new drug target.tuberculosis (tb) is still a major public health problem, compounded by the human immunodeficiency virus (hiv)-tb co-infection and recent emergence of multidrug-resistant (mdr) and extensively drug resistant (xdr)-tb. novel anti-tb drugs are urgently required. in this context, the 2c-methyl-d-erythritol 4-phosphate (mep) pathway of mycobacterium tuberculosis has drawn attention; it is one of several pathways vital for m. tuberculosis viability and the human host lacks homologous enzymes. thus, t ...200918793870
transcription inactivation through local refolding of the rna polymerase structure.structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. myxopyronin inhibits bacterial rna polymerase (rnap) by an unknown mechanism. here we report the structure of dmyx--a desmethyl derivative of myxopyronin b--complexed with a thermus thermophilus rnap holoenzyme. the antibiotic binds to a pocket deep inside the rnap clamp h ...200918946472
unfolding thermodynamics of the delta-domain in the prohead i subunit of phage hk97: determination by factor analysis of raman spectra.an early step in the morphogenesis of the double-stranded dna (dsdna) bacteriophage hk97 is the assembly of a precursor shell (prohead i) from 420 copies of a 384-residue subunit (gp5). although formation of prohead i requires direct participation of gp5 residues 2-103 (delta-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and dna packaging. the prohead i delta-domain is thought to resemble a phage scaffolding protein, by virtue of its highly alpha-helic ...200918983851
unfolding thermodynamics of the delta-domain in the prohead i subunit of phage hk97: determination by factor analysis of raman spectra.an early step in the morphogenesis of the double-stranded dna (dsdna) bacteriophage hk97 is the assembly of a precursor shell (prohead i) from 420 copies of a 384-residue subunit (gp5). although formation of prohead i requires direct participation of gp5 residues 2-103 (delta-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and dna packaging. the prohead i delta-domain is thought to resemble a phage scaffolding protein, by virtue of its highly alpha-helic ...200918983851
escherichia coli tmrna lacking pseudoknot 1 tags truncated proteins in vivo and in vitro.transfer-messenger rna (tmrna) and protein smpb facilitate trans-translation, a quality-control process that tags truncated proteins with short peptides recognized by a number of proteases and recycles ribosomes stalled at the 3' end of mrna templates lacking stop codons. the tmrna molecule is a hybrid of trna- and mrna-like domains that are usually connected by four pseudoknots (pk1-pk4). replacement of pk1 with a single-stranded rna yields pk1l, a mutant tmrna that tags truncated proteins very ...200919001120
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 ...200919017635
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 ...200919019162
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 ...200919020066
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 ...200919020066
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 ...200919022262
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 ...200919022262
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 ...200919028900
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 ...200919028900
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 ...200919029248
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 ...200919029248
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 ...200919029250
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 ...200919029250
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 ...200919036786
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 ...200919036786
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 ...200919036794
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 ...200919036794
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 ...200919015152
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 ...200919015152
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 ...200919015259
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 ...200919040641
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) ...200919059197
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) ...200919059197
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 ...200919063897
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 ...200919063897
frameshifting rna pseudoknots: structure and mechanism.programmed ribosomal frameshifting (prf) is one of the multiple translational recoding processes that fundamentally alters triplet decoding of the messenger rna by the elongating ribosome. the ability of the ribosome to change translational reading frames in the -1 direction (-1 prf) is employed by many positive strand rna viruses, including economically important plant viruses and many human pathogens, such as retroviruses, e.g., hiv-1, and coronaviruses, e.g., the causative agent of severe acu ...200918621088
frameshifting rna pseudoknots: structure and mechanism.programmed ribosomal frameshifting (prf) is one of the multiple translational recoding processes that fundamentally alters triplet decoding of the messenger rna by the elongating ribosome. the ability of the ribosome to change translational reading frames in the -1 direction (-1 prf) is employed by many positive strand rna viruses, including economically important plant viruses and many human pathogens, such as retroviruses, e.g., hiv-1, and coronaviruses, e.g., the causative agent of severe acu ...200918621088
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 ...200919166326
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 ...200919167482
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 ...200919171784
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 ...200919173642
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 ...200919176717
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 ...200919177357
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 ...200919177357
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.200919181809
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 ...200919182784
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 ...200919184529
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 ...200919186155
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 ...200919187032
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 ...200919187762
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 ...200919187763
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 ...200919188368
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 ...200919189973
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 ...200919190093
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 ...200919191548
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 ...200919193779
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. ...200919193993
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 ...200919193994
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.200919193996
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 ...200919194014
three genomes from the phylum acidobacteria provide insight into the lifestyles of these microorganisms in soils.the complete genomes of three strains from the phylum acidobacteria were compared. phylogenetic analysis placed them as a unique phylum. they share genomic traits with members of the proteobacteria, the cyanobacteria, and the fungi. the three strains appear to be versatile heterotrophs. genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. the genomes encode low-specificity major facilitator ...200919201974
three-dimensional structure of a1a0 atp synthase from the hyperthermophilic archaeon pyrococcus furiosus by electron microscopy.the archaeal atp synthase is a multisubunit complex that consists of a catalytic a(1) part and a transmembrane, ion translocation domain a(0). the a(1)a(0) complex from the hyperthermophile pyrococcus furiosus was isolated. mass analysis of the complex by laser-induced liquid bead ion desorption (lilbid) indicated a size of 730 +/- 10 kda. a three-dimensional map was generated by electron microscopy from negatively stained images. the map at a resolution of 2.3 nm shows the a(1) and a(0) domain, ...200919203996
characterization of dna polymerase x from thermus thermophilus hb8 reveals the polxc and php domains are both required for 3'-5' exonuclease activity.the x-family dna polymerases (polxs) comprise a highly conserved dna polymerase family found in all kingdoms. mammalian polxs are known to be involved in several dna-processing pathways including repair, but the cellular functions of bacterial polxs are less known. many bacterial polxs have a polymerase and histidinol phosphatase (php) domain at their c-termini in addition to a polx core (polxc) domain, and possess 3'-5' exonuclease activity. although both domains are highly conserved in bacteri ...200919211662
functional expression of a bacterial xylose isomerase in saccharomyces cerevisiae.in industrial fermentation processes, the yeast saccharomyces cerevisiae is commonly used for ethanol production. however, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. heterologous expression of a xylose isomerase (xi) would enable yeast cells to metabolize xylose. however, many attempts to express a prokaryotic xi with high activity in s. cerevisiae have failed so far. we have screened nucleic acid databases for sequences encoding putative xis and finally were a ...200919218403
characterization of a mimivirus rna cap guanine-n2 methyltransferase.a 2,2,7-trimethylguanosine (tmg) cap is a signature feature of eukaryal snrnas, telomerase rnas, and trans-spliced nematode mrnas. tmg and 2,7-dimethylguanosine (dmg) caps are also present on mrnas of two species of alphaviruses (positive strand rna viruses of the togaviridae family). it is presently not known how viral mrnas might acquire a hypermethylated cap. mimivirus, a giant dna virus that infects amoeba, encodes many putative enzymes and proteins implicated in rna transactions, including ...200919218551
reduction of hydrophilic ubiquinones by the flavin in mitochondrial nadh:ubiquinone oxidoreductase (complex i) and production of reactive oxygen species.nadh:ubiquinone oxidoreductase (complex i) from bovine heart mitochondria is a complicated, energy-transducing, membrane-bound enzyme that contains 45 different subunits, a non-covalently bound flavin mononucleotide, and eight iron-sulfur clusters. the mechanisms of nadh oxidation and intramolecular electron transfer by complex i are gradually being defined, but the mechanism linking ubiquinone reduction to proton translocation remains unknown. studies of ubiquinone reduction by isolated complex ...200919220002
a genetic screen for components of the mammalian rna interference pathway in bloom-deficient mouse embryonic stem cells.genetic screens performed in model organisms have helped identify key components of the rna interference (rnai) pathway. recessive genetic screens have recently become feasible through the use of mouse embryonic stem (es) cells that are bloom's syndrome protein (blm) deficient. here, we developed and performed a recessive genetic screen to identify components of the mammalian rnai pathway in blm-deficient es cells. genome-wide mutagenesis using a retroviral gene trap strategy resulted in the iso ...200919223321
dodecin is the key player in flavin homeostasis of archaea.flavins are employed to transform physical input into biological output signals. in this function, flavins catalyze a variety of light-induced reactions and redox processes. however, nature also provides flavoproteins with the ability to uncouple the mediation of signals. such proteins are the riboflavin-binding proteins (rfbps) with their function to store riboflavin for fast delivery of fmn and fad. here we present in vitro and in vivo data showing that the recently discovered archaeal dodecin ...200919224924
idiosyncratic helix-turn-helix motif in methanosarcina barkeri seryl-trna synthetase has a critical architectural role.all seryl-trna synthetases (serrss) are functional homodimers with a c-terminal active site domain typical for class ii aminoacyl-trna synthetases and an n-terminal domain involved in trna binding. the recently solved three-dimensional structure of methanosarcina barkeri serrs revealed the idiosyncratic features of methanogenic-type serrss; that is, an active site zinc ion, a unique trna binding domain, and an insertion of approximately 30 residues in the catalytic domain, which adopt a helix-tu ...200919228694
gtpase activation of elongation factor ef-tu by the ribosome during decoding.we have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the thermus thermophilus ribosome in which the ternary complex of elongation factor tu (ef-tu), trna and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. this represents the state in the decoding process just after codon recognition by trna and the resulting gtp hydrolysis by ef-tu, but before the release of ef-tu from the ribosome. progress in sample purificati ...200919229291
a molybdopterin oxidoreductase is involved in h2 oxidation in desulfovibrio desulfuricans g20.three mutants deficient in hydrogen/formate uptake were obtained through screening of a transposon mutant library containing 5,760 mutants of desulfovibrio desulfuricans g20. mutations were in the genes encoding the type i tetraheme cytochrome c(3) (cyca), fe hydrogenase (hydb), and molybdopterin oxidoreductase (mopb). mutations did not decrease the ability of cells to produce h(2) or formate during growth. complementation of the cyca and mopb mutants with a plasmid carrying the intact cyca and/ ...200919233927
stimulation of expression of a silica-induced protein (sip) in thermus thermophilus by supersaturated silicic acid.the effects of silicic acid on the growth of thermus thermophilus tmy, an extreme thermophile isolated from a siliceous deposit formed from geothermal water at a geothermal power plant in japan, were examined at 75 degrees c. at concentrations higher than the solubility of amorphous silica (400 to 700 ppm sio(2)), a silica-induced protein (sip) was isolated from the cell envelope fraction of log-phase tmy cells grown in the presence of supersaturated silicic acid. two-dimensional sodium dodecyl ...200919233950
domain architecture of the stator complex of the a1a0-atp synthase from thermoplasma acidophilum.a key structural element in the ion translocating f-, a-, and v-atpases is the peripheral stalk, an assembly of two polypeptides that provides a structural link between the atpase and ion channel domains. previously, we have characterized the peripheral stalk forming subunits e and h of the a-atpase from thermoplasma acidophilum and demonstrated that the two polypeptides interact to form a stable heterodimer with 1:1 stoichiometry (kish-trier, e., briere, l. k., dunn, s. d., and wilkens, s. (200 ...200919234304
improving the functional expression of a bacillus licheniformis laccase by random and site-directed mutagenesis.laccases have huge potential for biotechnological applications due to their broad substrate spectrum and wide range of reactions they are able to catalyze. these include, for example, the formation and degradation of dimers, oligomers, polymers, and ring cleavage as well as oxidation of aromatic compounds. potential applications of laccases include detoxification of industrial effluents, decolorization of textile dyes and the synthesis of natural products by, for instance, dimerization of phenol ...200919236694
structures of alternatively spliced isoforms of human ketohexokinase.a molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in western populations. fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (khk). here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic khk-c and the peripheral ...200919237742
dna uracil repair initiated by the archaeal exoiii homologue mth212 via direct strand incision.no genes for any of the known uracil dna glycosylases of the udg superfamily are present in the genome of methanothermobacter thermautotrophicus deltah, making it difficult to imagine how dna-u repair might be initiated in this organism. recently, mth212, the exoiii homologue of m. thermautotrophicus deltah has been characterized as a dna uridine endonuclease, which suggested the possibility of a novel endonucleolytic entry mechanism for dna uracil repair. with no system of genetic experimentati ...200919240141
involvement of a carboxylated lysine in uv damage endonuclease.uv damage endonuclease is a dna repair enzyme that can both recognize damage such as uv lesions and introduce a nick directly 5' to them. recently, the crystal structure of the enzyme from thermus thermophilus was solved. in the electron density map of this structure, unexplained density near the active site was observed at the tip of lys229. based on this finding, it was proposed that lys229 is post-translationally modified. in this article, we give evidence that this modification is a carboxyl ...200919241382
allopatric speciation in ticks: genetic and reproductive divergence between geographic strains of rhipicephalus (boophilus) microplus.the cattle tick, rhipicephalus (boophilus) microplus, economically impact cattle industry in tropical and subtropical regions of the world. the morphological and genetic differences among r. microplus strains have been documented in the literature, suggesting that biogeographical and ecological separation may have resulted in boophilid ticks from america/africa and those from australia being different species. to test the hypothesis of the presence of different boophilid species, herein we perfo ...200919243585
identification of neisserial dna binding components.neisseria meningitidis, a causative agent of meningitis and septicaemia, expresses type iv pili, a feature correlating with the uptake of exogenous dna from the environment by natural transformation. the outer membrane complex pilq, through which pili are extruded and retracted, has previously been shown to bind dna in its pore region. in order to further elucidate how dna is transported across the membranes, we searched for dna binding proteins within the meningococcal inner membrane. inner mem ...200919246756
the family x dna polymerase from deinococcus radiodurans adopts a non-standard extended conformation.deinococcus radiodurans is an extraordinarily radioresistant bacterium that is able to repair hundreds of radiation-induced double-stranded dna breaks. one of the players in this pathway is an x family dna polymerase (polx(dr)). deletion of polx(dr) has been shown to decrease the rate of repair of double-stranded dna breaks and increase cell sensitivity to gamma-rays. a 3'-->5' exonuclease activity that stops cutting close to dna loops has also been demonstrated. the present crystal structure of ...200919251692
crystallization and preliminary characterization of the thermus thermophilus rna helicase hera c-terminal domain.heat-resistant rna-dependent atpase (hera) from thermus thermophilus is a dead-box rna helicase. two constructs encompassing the second reca-like domain and the c-terminal domain of hera were overproduced in escherichia coli and purified to homogeneity. single crystals of both hera constructs were obtained in three crystal forms. a tetragonal crystal form belonged to space group p4(1)2(1)2, with unit-cell parameters a = 65.5, c = 153.0 a, and contained one molecule per asymmetric unit. two ortho ...200919255475
defects in transient trna translocation bypass trna synthetase quality control mechanisms.quality control mechanisms during protein synthesis are essential to fidelity and cell survival. leucyl-trna synthetase (leurs) misactivates non-leucine amino acids including isoleucine, methionine, and norvaline. to prevent translational errors, mischarged trna products are translocated 30a from the canonical aminoacylation core to a hydrolytic editing-active site within a completely separate domain. because it is transient, the trna translocation mechanism has been difficult to isolate. we hav ...200919258309
contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis.superoxide dismutase (sod) enzymes are critical in controlling levels of reactive oxygen species (ros) that are linked to aging, cancer, and neurodegenerative disease. superoxide (o(2)(*-)) produced during respiration is removed by the product of the sod2 gene, the homotetrameric manganese superoxide dismutase (mnsod). here, we examine the structural and catalytic roles of the highly conserved active-site residue tyr34, based upon structure-function studies of mnsod enzymes with mutations at thi ...200919265433
a novel endo-hydrogenase activity recycles hydrogen produced by nitrogen fixation.nitrogen (n(2)) fixation also yields hydrogen (h(2)) at 1:1 stoichiometric amounts. in aerobic diazotrophic (able to grow on n(2) as sole n-source) bacteria, orthodox respiratory hupsl-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen.200919277114
a recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center.the ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. here we demonstrate that the ribosomal peptidyl transferase center (ptc) is supported by a framework of magnesium microclusters (mg(2+)-muc's). common features of mg(2+)-muc's include two paired mg(2+) ions that are chelated by a common bridging phosphate group in the form mg((a))(2+)-(o1p-p-o2p)-mg((b))(2+). this bridging phosphate is part of a 10-membered chelation ring in t ...200919279186
an archaeal rad54 protein remodels dna and stimulates dna strand exchange by rada.rad54 protein is a key member of the rad52 epistasis group required for homologous recombination in eukaryotes. rad54 is a duplex dna translocase that remodels both dna and protein-dna complexes, and functions at multiple steps in the recombination process. here we use biochemical criteria to demonstrate the existence of this important protein in a prokaryotic organism. the sulfolobus solfataricus rad54 (ssorad54) protein is a double-strand dna-dependent atpase that can alter the topology of dup ...200919282450
the complete genome and proteome of laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats.laribacter hongkongensis is a newly discovered gram-negative bacillus of the neisseriaceae family associated with freshwater fish-borne gastroenteritis and traveler's diarrhea. the complete genome sequence of l. hongkongensis hlhk9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with g+c content of 62.35%. genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish ...200919283063
the genome of bacillus subtilis bacteriophage spo1.we report the genome sequence of bacillus subtilis phage spo1. the unique genome sequence is 132,562 bp long, and dna packaged in the virion (the chromosome) has a 13,185-bp terminal redundancy, giving a total of 145,747 bp. we predict 204 protein-coding genes and 5 trna genes, and we correlate these findings with the extensive body of investigations of spo1, including studies of the functions of the 61 previously defined genes and studies of the virion structure. sixty-nine percent of the encod ...200919285085
structural rearrangements in the active site of the thermus thermophilus 16s rrna methyltransferase ksga in a binary complex with 5'-methylthioadenosine.posttranscriptional modification of ribosomal rna (rrna) occurs in all kingdoms of life. the s-adenosyl-l-methionine-dependent methyltransferase ksga introduces the most highly conserved rrna modification, the dimethylation of a1518 and a1519 of 16s rrna. loss of this dimethylation confers resistance to the antibiotic kasugamycin. here, we report biochemical studies and high-resolution crystal structures of ksga from thermus thermophilus. methylation of 30s ribosomal subunits by t. thermophilus ...200919285505
resampling and editing of mischarged trna prior to translation elongation.faithful translation of the genetic code depends on the gtpase ef-tu delivering correctly charged aminoacyl-trnas to the ribosome for pairing with cognate codons. the accurate coupling of cognate amino acids and trnas by the aminoacyl-trna synthetases is achieved through a combination of substrate specificity and product editing. once released by aminoacyl-trna synthetases, both cognate and near-cognate aminoacyl-trnas were considered to be committed to ribosomal protein synthesis through their ...200919285947
structure and function of a campylobacter jejuni thioesterase cj0915, a hexameric hot dog fold enzyme.acyl-coenzyme a (coa) thioesterases are a large family of enzymes that hydrolyze acyl-coa esters to the free fatty acid and coa and thereby regulate essential cellular functions such as lipid metabolism, membrane synthesis, signal transduction, and gene transcription. to better understand the virulence mechanisms of campylobacter jejuni, and its possible link to membrane lipid biosynthesis, we have investigated c. jejuni thioesterases, annotated as putative proteins. while little is known about ...200919303060
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